BRCA1 associated polynucleotide (BAP-1) and uses therefor

ABSTRACT

Nucleic acid and amino acid sequences of a BRCA1 associated protein, BAP-1, are provided. These sequences, the protein, and anti-BAP-1 antibodies are useful in therapeutics and diagnostics for cancers associated with loss of the 3p21 chromosomal region and/or inappropriate BAP-1 levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC §371 of International Patent Application No. PCT/US97/13684, filed Jul. 30, 1997, which claims the benefit of the priority dates of U.S. Provisional Patent Application No. 60/038,109, filed Feb. 19, 1997, now abandoned and U.S. Provisional Patent Application No. 60/022,997, filed Aug. 2, 1996, now abandoned.

This invention was made under work supported by National Institutes of Health, Grant Nos. CA52009, DK49210, and TM54220. The United States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to the field of genes associated with cancers, and particularly, to BRCA1.

BACKGROUND OF THE INVENTION

The breast and ovarian cancer susceptibility gene, BRCA1, is linked to the hereditary form of breast cancer. The BRCA1 gene is located on chromosome 17 at the locus 17q21 and encodes a protein of 1863 amino acids. The BRCA 1 locus spans >100 kb comprising 24 exons [Miki et al, Science, 266:66-71 (1994)]. Expression of wild-type BRCA 1 inhibits colony function and tumor growth in vivo, whereas tumor derived mutations of BRCA 1 abolish this growth suppression [Holt et al, Nature Genetics, 12:298-302 (1996)]. Germline mutations in BRCA 1 appear to account for 50% of familial breast cancers and essentially all families with 17q21-linked inherited susceptibility to ovarian and breast cancer [Szabo et al, Hum. Mol. Genet., 4:1811-1817 (1995); Hall et al, Science, 250:1684-1689 (1990); Easton et al, Am. J. Hu. Genet., 56:265-271 (1995); Narod et al, Am. J. Hu. Genet., 56:254-264 (1995)]. Kindreds segregating constitutive BRCA1 mutations show a lifetime risk of 40-50% for ovarian cancer and >80% for breast cancer [Easton et al, Am. J. Hum. Genet., 52:678-701 (1993); Easton et al, Am. J. Hum. Genet., 56:265-271 (1995)].

The classification of BRCA 1 as a highly penetrant, autosomal dominant tumor suppressor gene has been genetically confirmed by the finding of frequent loss or mutation (LOH) of the wild-type allele in breast tumors from mutation carriers [Hall et al, Science, 250:1684-1689 (1990); Miki et al, cited above; Smith et al, Nature Genetics, 2:128-131 (1992)]. Surprisingly, BRCA 1 mutations in sporadic breast cancer including those which show LOH have yet to be found and are extremely rare in sporadic ovarian cancer [Futreal et al, Science, 266:120-122 (1994); Merajver et al, Nature Genetics, 9:439-443 (1995)].

Although the BRCA1 protein resembles no known protein, it does contain a RING domain at its amino terminus [Miki, cited above; Bienstock et al, Cancer Res., 56:2539-2545 (1996)]. The RING finger domain is a complicated structure which chelates two zinc atoms using 7 Cys residues and 1 His residue [C₃HC₄; Lovering et al, Proc. Natl. Acad. Sci. USA, 90:2112-2116 (1993); Freemont et al, Ann. NY Acad. Sci., 684:174-192 (1993)]. This domain is present in a wide variety of proteins with various functions, but the function of the RING finger domain within these proteins is unknown [for a review see Saurin, Trends in Biochem. Sci., 21:208-214 (1996)]. The RING finger of BRCA1 is important to its function since missense mutations in the RING domain (Cys61Gly and Cys64Gly) are found in breast/ovarian kindreds [Friedman et al, Nat. Genet., 8:399-404 (1994); Merajver, cited above; Castilla et al, Nature Genet., 8:387-391 (1994)]. In addition, the RING finger domain is the most conserved region of BRCA1, when comparing the human, mouse and rat proteins. The BRCA1 RING finger is anticipated to be a binding site for protein(s) which either mediate BRCA1 tumor suppressor function or serve to regulate these functions. Genetic evidence supports this in that single amino-acid substitutions at metal chelating cysteines, C61G and C64G, occur in kindreds; these mutations segregate with the disease susceptibility phenotype and are predicted to abolish RING finger structure.

Other functions of BRCA1 are discussed in the following references which are incorporated herein by reference: Borden et al, EMBO J., 14:1532-1541 (1995); Lovering et al, Proc. Natl. Acad. Sci. USA, 90:2112-2116 (1993); Koonin et al, Nature Genet., 13:266-268 (1996); Chen et al, Science, 270:789-791 (1995); Chen et al, Cancer Research, 56:3168-3172 (1996); Scully et al, Science, 272:123-126 (1996); Thakur et al, Molecular & Cellular Biology, 17:444-452 (1997); Scully et al, Cell, 88:265-275 (1997); Chapman et al, Nature, 382:678-679 (1996); Scully et al, Proc. Natl. Acad. Sci. USA, 94:5605-5610 (1997); Marquis et al, Nature Genetics, 11:17-26 (1995); Gudas et al, Cancer Res., 55:4561-4565 (1995); Gudas et al, Cell Growth and Differentiation, 7:717-723 (1996); Vaughn et al, Cell Growth and Differentiation, 7:711-715 (1996); Marks et al, Oncogene, 14:115-121 (1997); Zabludoff et al, Oncogene, 13:649-653 (1996); Hakem et al, Cell, 85:1009-1023 (1996); Liu et al, Genes & Development, 10:1835-1843 (1996); Rao et al, Oncogene, 12:523-528 (1996); Thompson et al, Nature Genetics, 9:444-450 (1995); Chen et al, J. Biol. Chem., 271:32863-32868 (1996); Wu et al, Nature Genetics, 14:430-440 (1996); Klug et al, FASEB Journal, 9:597-604 (1995); Saurin et al, Trends in Biochem. Sci., 21:208-214 (1996); Friedman et al, Genes & Development, 10:2067-2078 (1996); Neuhausen & Marshall, Cancer Res., 54:6069-6072 (1994); Schildkraut et al, Am. J. Obstet. Gynecol., 172:908-913 (1995); FitzGerald et al, N. Engl. J. Med., 334:143-149 (1996); Ford et al, Lancet, 343:692-695 (1994); Muto et al, Cancer Research, 56:1250-1252 (1996); Rao et al, Nature Genetics, 14:185-187 (1996), Struewing et al, Nature Genetics, 11:198-200 (1995); Couch et al, Human Mutation, 8:8-18 (1996); Holt et al, Nature Genetics, 12:298-302 (1996); Jensen et al, Nature Genetics, 12:303-308 (1996); Bradley & Sharan, Nature Genetics, 13:268-271 (1996).

There is a need in the art for compositions and methods useful in the treatment and/or prophylaxis of cancers caused by loss of, and mutations in, BRCA1.

SUMMARY OF THE INVENTION

The present invention meets the needs in the art by identifying a novel mammalian BRCA1 Associated Protein (BAP-1) and nucleic acid sequences encoding same. BAP1 is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified and is a new tumor suppressor gene which functions in the BRCA1 growth control pathway. Compositions, both diagnostic and therapeutic, based on this newly identified protein are provided herein.

Thus, in one aspect, the present invention provides a nucleic acid sequence, which is isolated from cellular materials with which it is naturally associated. The nucleic acid sequence is preferably selected from SEQ ID NO:1, or a fragment thereof. Such a fragment may have a specified biological function as discussed below, or may encode a peptide having a similar biological function as the intact BAP-1. Homologous nucleotide sequences, and modified nucleotide sequences which encode peptides or proteins which have a similar biological function as the intact BAP-1, are also included in this aspect of the invention.

In another aspect, the present invention provides a mammalian BRCA1 associated protein (BAP-1). In one preferred embodiment, the protein is human and has the amino acid sequence of SEQ ID NO:2. In another embodiment a fragment of the SEQ ID NO:2 encodes a peptide having a similar biological function as the intact BAP-1 protein. Amino acid sequences homologous to SEQ ID NO: 2, and modified amino acid sequences of SEQ ID NO: 2, which encode peptides or proteins which have a similar biological function as the intact BAP-1 or a specified biological function as discussed below, are also included in this aspect of the invention.

In yet another aspect, the present invention provides a polynucleotide molecule, for example, a vector or plasmid, that comprises a mammalian BAP-1 nucleic acid sequence as defined herein under the control of suitable sequences which direct and regulate expression of the BAP-1 nucleic acid sequence.

In a further aspect, the present invention provides a host cell transformed with a polynucleotide molecule or vector of the invention.

In yet a further aspect, the present invention provides a method of recombinantly expressing BAP-1 or a peptide fragment thereof, by culturing a recombinant host cell according to the invention under conditions which permit expression of BAP-1 or a fragment thereof.

In still a further aspect, the present invention provides an anti-BRCA1 associated protein (BAP-1) antibody.

In yet another aspect, the invention provides a diagnostic reagent comprising an antibody of the invention and a detectable label. Alternatively, a diagnostic reagent of the invention may comprise a nucleic acid sequence of the invention, or a fragment thereof, and a detectable label which is associated with said sequence.

In still another aspect, the invention provides a method of detecting a cancer associated with abnormal levels of BAP-1 comprising providing a biopsy sample from a patient suspected of having said cancer and incubating said sample in the presence of a diagnostic reagent of the invention.

In a further aspect, the present invention provides methods of identifying compounds which specifically bind to BAP-1 or a fragment thereof. In still a further aspect, the present invention provides for compounds or drugs produced by use of the above methods.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRITION OF THE DRAWINGS

FIG. 1A illustrates the structural features of the BRCA1 gene product. It shows an alignment of RING finger domains of human BRCA1 [SEQ ID NO:3] and mouse BRCA1 [SEQ ID NO:4] (AA1-100), RPT-1 (amino acids 12-100 of SEQ ID NO:5), a putative lymphocyte specific transcription factor having the most closely related RING finger, and BARD1 (AA 47-89) [SEQ ID NO: 19]. Asterisks (*) identify the Zn-chelating amino acids that form the core of the RING finger. Boxed amino acids show regions of identity between the RING finger domains of human BRCA1 and the other proteins. Alignment was performed by ClustalW [Thompson et al, Nucleic Acids Research, 22:4673-4680 (1994)].

FIG. 1B is a schematic map which illustrates the constructs made when the amino terminal 100 amino acids of human BRCA1 (which includes the RING finger domain) and the indicated amino acids of the various BRCA1-RF mutants and controls (described in Example 1) were fused to the LexA DNA-binding domain. The signature C3HC4 structure is highlighted.

FIG. 2A provides a comparison of the amino terminal regions of BAP-1 (FLBAP) [amino acids 1-257 of SEQ ID NO: 2], a C. elegans 3 protein [SEQ ID NO: 16], and human ubiquitin carboxyl-terminal hydrolase isozymes L1 (human UBL1) [SEQ ID NO: 17] and L3 (human UBL3) [SEQ ID NO: 18]. Boxed regions indicate areas of greater than 85% homology. This region contains the active sites of UBL1 and UBL3.

FIG. 2B illustrates the sequences and provides a comparison of the partial human [SEQ ID NO: 6] and mouse BAP-1 proteins [SEQ ID NO: 7-9] isolated via the yeast 2-hybrid screens of Example 2. Capital letters encode BAP-1. Lower case letters represent the amino acids encoded by the vector. Human BAP-1 is fused to Gal4 activation domain. Mouse BAP-1 is fused to the VP16 activation domain.

FIGS. 3A-3E provide the nucleic acid [SEQ ID NO:1] and amino acid [SEQ ID NO:2] sequences of the novel ubiquitin carboxy-terminal hydrolase, BAP-1. The longest open reading frame which contained the amino acids defined by the (human) 2-hybrid fusion protein is 2188 nucleotides encoding 729 amino acids. The cDNA also contains 39 nucleotides of 5′UTR and 1705 nucleotides of 3′UTR. The enzymatic active site is contained within the first 250 amino acids; the active site residues are circled. The putative nuclear localization signals (NLS) are underlined, the highly acidic region is boxed with heavy lines, the interaction domain is boxed and the protein fragment used to generate BAP1 polyclonal antibodies is bracketed (A.A.'s 483-576 of SEQ ID NO: 2). The conserved amino acids of the ubiquitin COOH-terminal hydrolase active site consensus are circled (amino acids 91, 169, 184 of SEQ ID NO: 2).

FIG. 3F is a comparison of BAP1 (amino acids 1-261 of SEQ ID NO: 2) with other UCH's. UCH-CAEEL (genbank #Q09444) (amino acids 1-251 of SEQ ID NO: 20), UCH DROME (genbank #P35122) [SEQ ID NO: 21] (aa 1-227), YUH1 (genbank #P35127) [SEQ ID NO: 22] (aa 1-236), UCHL-1 (genbank #P09936) [SEQ ID NO: 24] (aa 1-223), UCHL-3 (genbank #P15374) [SEQ ID NO: 23] (aa 1-230). BAP1 (amino acids 630-729 of SEQ ID NO: 2) is further compared to CAEEL-CO8B11.7 (amino acids 238-326 of SEQ ID NO: 20). The BLAST search algorithm was used to identify proteins closely related to BAP1 [Altschul et al, J. Mol. Biol., 215:403-410 (1990)]. The UCH domain of four of these proteins were aligned with BAP1 using the CLUSTALW (ver.1.6) algorithm [Thompson et al, cited above]. Areas of homology with other UCH's are boxed. Only CAEEL-CO8B11.7 showed any homology outside of the enzymatic region.

FIG. 3G is a schematic comparison of the BAP1 and UCH's. The region necessary for the interaction with BRCA1 (AAs 598-729) is indicated in the diagrams with light crosshatching.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein, BRCA1 associated protein-1 (BAP1). BAP1 is a novel, nuclear localized, enzyme which displays the signature motifs and activities of a ubiquitin carboxy-terminal hydrolase, i.e., BAP1 cleaves model ubiquitin substrates in vitro. In fact, BAP1 is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. The ubiquitin hydrolase function of BAP1 implicates the ubiguitin-proteasome pathway in either the regulation, or as a direct effector, of BRCA1 function. Thus, BAP1 likely has a broad role in ubiquitin-dependent regulatory processes within the nucleus, including the emerging role of ubiquitin conjugation as a subcellular targeting signal, as well as in transcription, chromatin remodeling, cell cycle control and DNA repair/recombination.

BAP1 also enhances the tumor growth suppression properties of BRCA1 in colony formation assays and does so in a manner dependent upon the UCH enzymatic domain and the BRCA1-interaction domain. BAP1 specifically binds to the wild-type BRCA1 RING finger domain (BRCA1-RF) both in vitro and in vivo, but not to mutant BRCA1-RF's, e.g., the C61G or C64G mutated RING fingers found in tumors from breast cancer kindreds or other closely related RING fingers. The interaction between BAP1 and BRCA1 occurs in vitro and BAP1 mRNA is expressed in those tissues which also express BRCA1. Thus, BAP1 has a role as a tumor suppressor gene.

As described below, the yeast two-hybrid system was employed to isolate mouse and human clones of BAP1. The human BAP1 locus was mapped to human chromosome 3p21.3. Rearrangements and intragenic homozygous deletions and mutations of BAP1 have been found in lung carcinomas, including homozygous deletions found in non-small cell lung cancers.

Together, this evidence supports the role of BAP1 as a tumor suppressor gene and as a regulator or an effector in BRCA1 growth control pathways. Both the specificity of the BRCA1 RING finger-BAP1 interaction and the fact that independent, tumor-derived missense mutations in the cysteines in the BRCA1 RING finger domain abolish interaction with BAP1 provide compelling evidence for the physiological relevance of this interaction.

The invention further provides nucleic acid sequences which encode BAP1 or fragments of BAP1 which have a biological function, diagnostic and therapeutic reagents, as well as methods of using BAP1, its nucleic acid sequences, and antibodies developed thereto. The nucleic acid sequences, protein, amino acid sequences and antibodies directed to BAP1 are useful in the detection, diagnosis and treatment of cancers associated with inappropriate BAP-1 levels and/or loss of chromosomal region 3p21.

In one embodiment, the nucleic acid sequence of the invention is an about 3.5 kb cDNA [SEQ ID NO: 1], encoding BAP-1. BAP-1 is a 729 amino acid protein [SEQ ID NO: 2] which interacts through its carboxy terminus with the BRCA1 RING finger domain. In addition to containing the 250 amino acid amino terminal UCH catalytic domain, it includes a long carboxy-terminal extension with rich in proline, serine and threonine and and contains a short region of extreme acidity in which 12 of 13 amino acids are either Glu or Asp, elements which may confer a short half-life upon the protein [Rechsteiner et al, Trends Biochem. Sci., 21:267-271 (1996)]. The extreme carboxy-terminus encodes two potential nuclear localization signals which overlap the approximately 125 amino acid BRCA1-interaction domain. It was this domain that was independently isolated from mouse and human libraries in the two-hybrid screen of Example 2 and is predicted to fold into a long amphipathic helix of coiled-coil character, the structure of which may be important for BRCA1 interaction. Truncation into this region or substitution of a proline for leucine 691 abolish the BAP1-BRCA1 interaction. A potential splice variant in BAP1 results in loss of 31 amino acids of the BRCA1 interaction domain and greatly reduces the ability of BAP1 to bind the BRCA1 RING finger, further suggesting that the BAP1-BRCA1 interaction is regulated. Thus, the BAP1 carboxy-terminus is tethered to BRCA1 via the RING finger domain and that the UCH catalytic domain is free to interact with ubiquitin substrates.

Northern analysis showed that BAP-1 is a ^(˜)4 kb mRNA expressed in a variety of tissues and cell lines. The cDNA encodes a protein of 80 kD predicted molecular weight. However, expression of the cDNA in vitro or in COS1 cells generated a protein with an apparent molecular weight of approximately 91 kDa suggesting possible post-translational modifications. Localization of BAP-1 by cell fractionation indicated that it is predominantly a nuclear protein. Chromosomal analysis by fluorescent in situ hybridization (FISH) localized BAP-1 to chromosome 3p21, a genomic region found to be deleted in some breast cancers. The loss of BAP-1 function, individually or in tandem with BRCA1, is anticipated to be associated with breast cancer progression. Thus, the BAP-1 protein may mediate BRCA1 function and inhibit its oncogenic activity. These and other aspects of the invention are discussed in more detail below.

BRCA1 is likely a direct substrate for the UCH activity of BAP1. Thus, in contrast to all of the known UCHs which are comprised entirely of the UCH domain, the carboxy-terminal extension of BAP1 provides substrate and/or targeting specificity for the catalytic function. Regulated ubiquitination of BRCA1 and subsequent proteasome-mediated degradation would not be surprising given that both BRCA1 levels and subnuclear localization are tightly regulated in the mitotic cell-cycle and during meiosis [Gudas et al, cited above; Scully et al, cited above; Zabludoff et al, cited above]. BAP1-mediated deubiquitination of BRCA1 would be expected to stabilize the protein and protect it from proteasome-mediated degradation. This is consistent with both the ability of co-transfected BAP1 to enhance the tumor suppressor effects of BRCA1 in colony formation assays and the finding of mutations in BAP1 in cancer cell lines.

The BRCA1-BAP1 association may also serve to target the UCH domain to other substrates. These substrates may be bound to other sites on BRCA1. BRCA1 could be construed as an assembly or scaffold molecule for regulated assembly of multiprotein complexes, a function which has been postulated for other tumor suppressor proteins [e.g. pRb; Sellers et al, Biochim. Biophys. Acta., 1288:M1-5 (1996); Welch et al, Genes Dev., 9:31-46 (1995)]. BAP1 may thus be a regulator of this assembly via controlled ubiquitin proteolysis, similar to two other RING finger-containing proteins involved in controlled proteolysis processes, i.e., a mouse homologue of the drosophila seven-in-absentia (siah; a RING finger protein) and the herpes virus protein VMW110 RING finger protein [Everett et al, EMBO J., 16:566-577 (1997)].

The following description defines the aspects of this invention in more detail.

I. Nucleic Acid Sequences

The present invention provides mammalian nucleic acid sequences encoding BAP-1. The nucleic acid sequences of this invention are isolated from cellular materials with which they are naturally associated. In one embodiment, a BAP-1 cDNA sequence is provided in SEQ ID NO:1 (FIGS. 3A-3E).

Given the cDNA sequences of SEQ ID NO: 1, one of skill in the art can readily obtain the corresponding anti-sense strands of these DNA sequences. Further, using known techniques, one of skill in the art can readily obtain genomic sequences corresponding to these DNA sequences or the corresponding RNA sequences, as desired.

Similarly the availability of SEQ ID NO: 1 of this invention permits one of skill in the art to obtain other species BAP-1 analogs, by use of the nucleic acid sequences of this invention as probes in a conventional technique, e.g., polymerase chain reaction. Allelic variants of these sequences within a species (i.e., sequences containing some individual nucleotide differences from a more commonly occurring sequence within a species, but which nevertheless encode the same protein) such as other human variants of BAP-1 SEQ ID NO: 2, may also be readily obtained given the knowledge of his sequence provided by this invention.

The present invention further encompasses nucleic acid sequences capable of hybridizing under stringent conditions [see, J. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory (1989)] to the sequences of SEQ ID NO: 1, their anti-sense strands, or biologically active fragments thereof. An example of a highly stringent hybridization condition is hybridization at 2×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for an hour. Alternatively, an exemplary highly stringent hybridization condition is in 50% formamide, 4×SSC at 42° C. Moderately high stringency conditions may also prove useful, e.g. hybridization in 4×SSC at 55° C., followed by washing in 0.1×SSC at 37° C. for an hour. An alternative exemplary moderately high stringency hybridization condition is in 50% formamide, 4×SSC at 30° C.

Also encompassed within this invention are fragments of the above-identified nucleic acid sequences. Preferably, such fragments are characterized by encoding a biologically active portion of BAP-1, e.g., an epitope. Generally, these oligonucleotide fragments are at least 15 nucleotides in length. However, oligonucleotide fragments of varying sizes may be selected as desired. Such fragments may be used for such purposes as performing the PCR, e.g., on a biopsied tissue sample. For example, particularly useful fragments of BAP-1 cDNA [SEQ ID NO:1 ] and corresponding sequences include the open reading frame, nt 40-2226, the nuclear localization sites, nt 2005 to 2022 and nt 2188 to 2205, a region of acidity at nt 1225 to 1263, and the BRCA1-RF-interactive domain at nt 1831 to 2226 of SEQ ID NO:1. Other fragments which are contained within the above identified fragments or which overlap them and demonstrate similar biological activities, e.g., those which differ by 1 to 9 bases, are also desirable. Similarly, other useful fragments may be readily identified by one of skill in the art by resort to conventional techniques, such as, by deletion mutagenesis, fusion to other proteins, or by motif searches in computer databases. In addition, other suitable techniques are known.

The nucleotide sequences of the invention may be isolated by conventional uses of polymerase chain reaction or cloning techniques such as those described in obtaining the murine and human sequences, described below. Alternatively, these sequences may be constructed using conventional genetic engineering or chemical synthesis techniques.

According to the invention, the nucleic acid sequences may be modified. Utilizing the sequence data in FIGS. 3A-3E [SEQ ID NO: 1] and in the sequence listing, it is within the skill of the art to obtain other polynucleotide sequences encoding the proteins of the invention. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion. Also included are allelic variations, caused by the natural degeneracy of the genetic code.

Also encompassed by the present invention are mutants of the BAP-1 gene provided herein. Such mutants include amino terminal, carboxy terminal or internal deletions which are useful as dominant inhibitor genes. Such a truncated, or deletion, mutant may be expressed for the purpose of inhibiting the activity of the full-length or wild-type gene.

These nucleic acid sequences are useful for a variety of diagnostic and therapeutic uses. Advantageously, the nucleic acid sequences are useful in the development of diagnostic probes and antisense probes for use in the detection and diagnosis of conditions characterized by BRCA1 mutation. Additionally, the BAP-1 gene has been mapped to chromosome 3p21.3. Thus, these sequences provide a good marker for further analysis of chromosome 3. The nucleic acid sequences of this invention are also useful in the production of mammalian, and particularly, human BAP-1 proteins and peptides.

II. Protein Sequences

The present invention also provides mammalian BAP-1 polypeptides, peptides or proteins. These proteins are free from association with other contaminating proteins or materials with which they are found in nature. In one embodiment, the invention provides a human BAP-1 [SEQ ID NO:2] polypeptide of 729 amino acids having a predicted molecular weight (MW) of about 81 kD. In another embodiment, the invention provides partial human and murine BAP-1 proteins [SEQ ID NO: 6-9] (FIG. 2B).

Also included in the invention are analogs, or modified versions, of the proteins provided herein. Typically, such analogs differ by only one to four codon changes. Examples include polypeptides with minor amino acid variations from the illustrated amino acid sequences of BAP-1 (FIGS. 3A-3E) [SEQ ID NO: 2]; in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Also provided are homologs of the proteins of the invention which are characterized by having at least about 85% or higher homology with SEQ ID NO:2. Based on the sequence information provided herein, one of skill in the art can readily obtain BAP-1 from other mammalian species.

Further encompassed by this invention are fragments of the BAP-1 polypeptide. Such fragments are desirably characterized by having BAP-1 biological activity, including, e.g., the ability to bind specifically to the RING finger domain of wild-type BRCA1. These fragments may be designed or obtained in any desired length, including as small as about 5-8 amino acids in length. Such a fragment may represent an epitope of the protein. Alternatively, the BAP-1 proteins [SEQ ID NO:2] of the invention may be modified, for example, by truncation at the amino or carboxy termini, by elimination or substitution of one or more amino acids, or by any number of now conventional techniques to improve production thereof, to enhance protein stability or other characteristics, e.g. binding activity or bioavailability, or to confer some other desired property upon the protein.

Currently, desirable proteins or peptides correspond to the nuclear localization sites, residues 656 to 661 and residues 717 to 722 of SEQ ID NO:2, a region of extreme acidity, residues 396 to 408 SEQ ID NO:2, and the interactive domain, residues 598 to 729 of SEQ ID NO:2. Another suitable fragment, which has homology to ubiquitin carboxyl-terminal hydrolase, isozyme L3, is located between about amino acids 1 to about 214 of SEQ ID NO: 2. Yet another suitable fragment, corresponding to residues 483 to 576 of SEQ ID NO:2 has been used to generate antibodies. Other suitable fragments include amino acids 1 to 313, 1 to 325, 1 to 352, and 1 to 426 of SEQ ID NO: 2. Additionally, fragments which are about in the range of the above amino acid residues, e.g., which differ by 1 to 5 amino acids, are anticipated to be particularly desirable. Still other suitable BAP-1 fragments are identified in the Examples or may be readily identified and prepared by one of skill in the art using known techniques, such as deletion mutagenesis and expression.

III. Expression

A. In Vitro

To produce recombinant BAP-1 proteins of this invention, the DNA sequences of the invention are inserted into a suitable expression system. Desirably, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding BAP-1 is operably linked to a heterologous expression control sequence permitting expression of the BAP-1 protein. Numerous types of appropriate expression vectors are known in the art for mammalian (including human) protein expression, by standard molecular biology techniques. Such vectors may be selected from among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose.

Methods for obtaining such expression vectors are well-known. See, Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New York (1989); Miller et al, Genetic Engineerin, 8:277-298 (Plenum Press 1986) and references cited therein.

Suitable host cells or cell lines for transfection by this method include mammalian cells, such as Human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice may be used. Another suitable mammalian cell line is the CV-1 cell line. Still other suitable mammalian host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art. [See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446].

Similarly bacterial cells are useful as host cells for the present invention. For example, the various strains of E. coli (e.g., HB101, MC1061, and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Other fungal cells may also be employed as expression systems.

Alternatively, insect cells such as Spodoptera frugipedera (Sf9) cells may be used.

Thus, the present invention provides a method for producing a recombinant BAP-1 protein which involves transfecting a host cell with at least one expression vector containing a polynucleotide of the invention under the control of a transcriptional regulatory sequence, e.g., by conventional means such as electroporation. The transfected or transformed host cell is then cultured under conditions that allow expression of the BAP-1 protein. The expressed protein may then be recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art.

For example, the proteins may be isolated in soluble form following cell lysis, or may be extracted using known techniques, e.g., in guanidine chloride. If desired, the BAP-1 proteins of the invention may be produced as a fusion protein. For example, it may be desirable to produce BAP-1 fusion proteins, to enhance expression of the protein in a selected host cell, to improve purification, or for use in monitoring the presence of BAP-1 in tissues, cells or cell extracts. Suitable fusion partners for the BAP-1 proteins of the invention are well known to those of skill in the art and include, among others, β-galactosidase, glutathione-S-transferase, and poly-histidine.

B. In Vivo

Alternatively, where it is desired that the BAP-1 protein (whether full-length or a desirable fragment) be expressed in vivo, e.g., for gene therapy purposes, an appropriate vector for delivery may be readily selected by one of skill in the art. Exemplary gene therapy vectors are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus [International patent application No. PCT/US91/03440], adenovirus vectors [M. Kay et al, Proc. Natl. Acad. Sci. USA, 91:2353 (1994); S. Ishibashi et al, J. Clin. Invest., 92:883 (1993)], or other viral vectors, e.g., various poxviruses, vaccinia, etc. Methods for insertion of a desired gene, e.g., BAP-1, and obtaining in vivo expression of the encoded protein, are well known to those of skill in the art.

IV. Antisera and Antibodies

The BAP-1 proteins of this invention are also useful as antigens for the development of anti-BAP-1 antisera and antibodies to BAP-1 or to a desired fragment of a BAP-1 protein. Specific antisera may be generated using known techniques. See, Sambrook, cited above, Chapter 18, generally, incorporated by reference and Example 5 below. Similarly, antibodies of the invention, both polyclonal and monoclonal, may be produced by conventional methods. These techniques may include the Kohler and Milstein hybridoma technique, recombinant techniques, such as described by Huse et al, Science, 246:1275-1281 (1988), or any other techniques known to the art.

Also encompassed within this invention are humanized and chimeric antibodies. As used herein, a humanized antibody is defined as an antibody containing murine complementary determining regions (CDRS) capable of binding to BAP-1 or a fragment thereof, and human framework regions. These CDRs are preferably derived from a murine monoclonal antibody (MAb) of the invention. As defined herein, a chimeric antibody is defined as an antibody containing the variable region light and heavy chains, including both CDR and framework regions, from a BAP-1 MAb of the invention and the constant region light and heavy chains from a human antibody. Methods of identifying suitable human framework regions and modifying a MAb of the invention to contain same to produce a humanized or chimeric antibody of the invention, are well known to those of skill in the art. See, e.g., E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994). Other types of recombinantly-designed antibodies are also encompassed by this invention.

Further provided by the present invention are anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for the target to which anti-BAP-1 antibodies of the invention bind and Ab3 are similar to BAP-1 antibodies (Ab1) in their binding specificities and biological activities [see, e.g., M. Wettendorff et al., “Modulation of anti-tumor immunity by anti-idiotypic antibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux J, Am. Soc. Microbiol., Washington D.C.: pp. 203-229, (1990)]. These anti-idiotype and anti-anti-idiotype antibodies may be produced using techniques well known to those of skill in the art. Such anti-idiotype antibodies (Ab2) can bear the internal image of BAP-1 and bind to BRCA1 in much the same manner as BAP-1, and are thus useful for the same purposes as BAP-1.

In general, polyclonal antisera, monoclonal antibodies and other antibodies which bind to BAP-1 as the antigen (Ab1) are useful to identify epitopes of BAP-1, to separate BAP-1 from contaminants in living tissue (e.g., in chromatographic columns and the like), and in general as research tools and as starting material essential for the development of other types of antibodies described above. Anti-idiotype antibodies (Ab2) are useful for binding BRCA1 and thus may be used in the treatment of cancers. The Ab3 antibodies may be useful for the same reason the Ab1 are useful. Other uses as research tools and as components for separation of BAP-1 from other contaminant of living tissue, for example, are also contemplated for the above-described antibodies.

V. Diagnostic Reagents and Methods

Advantageously, the present invention provides reagents and methods useful in detecting and diagnosing abnormal levels of BAP-1, (i.e., deficiencies or excesses thereof) in a patient. Conditions associated with excess levels of BAP-1 may be indicative of BRCA1 mutations. Abnormal levels of BAP-1 may be associated with a variety of cancers, including lung cancer (small cell lung carcinoma and non-small cell lung carcinoma), breast cancers, uterine carcinomas, and oral squamous cell carcinomas, among others.

Thus, the proteins, protein fragments, antibodies, and polynucleotide sequences (including anti-sense polynucleotide sequences and oligonucleotide fragments), and BAP-1 antisera and antibodies of this invention may be useful as diagnostic reagents. These reagents may optionally be labelled using diagnostic labels, such as radioactive labels, calorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods. Alternatively, the N- or C-terminus of BAP-1 or a fragment thereof may be tagged with a viral epitope which can be recognized by a specific antisera. The reagents may be used to measure abnormal BAP-1 levels in selected mammalian tissue using conventional diagnostic assays, e.g., Southern blotting, Northern and Western blotting, polymerase chain reaction (PCR), reverse transcriptase (RT) PCR, immunostaining, and the like. For example, in biopsies of tumor tissue, loss of BAP-1 expression in tumor tissue could be directly verified by RT-PCR or immunostaining. Alternatively, a Southern analysis, genomic PCR, or fluorescence in situ hybridization (FISH) may be performed to confirm BAP-1 gene rearrangement.

In one example, as diagnostic agents the polynucleotide sequences may be employed to detect or quantitate normal BAP-1. The selection of the appropriate assay format and label system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.

Thus the present invention provides methods for the detection of disorders characterized by inappropriate BAP-1 levels. The protein, antibody, antisera and polynucleotide reagents of the invention are expected to be useful in the following methods. The methods involve contacting a selected mammalian tissue, e.g., a biopsy sample or other cells, with the selected reagent, protein, antisera antibody or DNA sequence, and measuring or detecting the amount of BAP-1 present in the tissue in a selected assay format based on the binding or hybridization of the reagent to the tissue.

VI. Therapeutic Compositions and Methods

BAP-1 is believed to have a role in modulating the activity of BRCA1, a tumor suppressor. More particularly, BAP-1 enzymatic activity is anticipated to have a role in the persistence of BRCA1 in a cell. For example, the extended presence of BRCA1, particularly in high levels, is associated with cell death. Thus, by adjusting BAP-1 levels in a cell, e.g., by use of BAP-1 or an inhibitor identified by the invention, persistence of BRCA-1 in the cells can thereby be altered. For example, it may be desirable to adjust BAP-1 levels so as to enhance BRCA1 persistence in a cell, e.g., a tumor cell. Alternatively, it may be desirable to adjust BAP-1 levels so as to increase BRCA1 degradation in the cell. The compositions and methods useful for the treatment of conditions associated with inadequate or undesirable BAP-1 levels are provided. As stated above, included among such conditions are liver and breast cancers.

The therapeutic compositions of the invention may be formulated to contain an anti-idiotype antibody of the invention, the BAP-1 protein itself or a fragment thereof. The therapeutic composition desirably contains 0.01 μg to 10 mg protein. These compositions may contain a pharmaceutically acceptable carrier. Suitable carriers are well known to those of skill in the art and include, for example, saline. Alternatively, such compositions may include conventional delivery systems into which protein of the invention is incorporated. Optionally, these compositions may contain other active ingredients, e.g., chemotherapeutics.

Still another method involves the use of the BAP-1 polynucleotide sequences for gene therapy. In the method, the BAP-1 sequences are introduced into a suitable vector for delivery to a cell containing a deficiency of BAP-1 and/or to block tumor growth. By conventional genetic engineering techniques, the BAP-1 gene sequence may be introduced to mutate the existing gene by recombination or to replace an inactive or missing gene.

Generally, a suitable vector-based treatment contains between 1×10⁻³ pfu to 1×10⁻¹² pfu per dose. However, the dose, timing and mode of administration of these compositions may be determined by one of skill in the art. Such factors as the age, condition, and the level of the BAP-1 deficiency detected by the diagnostic methods described above, may be taken into account in determining the dose, timing and mode of administration of the therapeutic compositions of the invention. Generally, where treatment of an existing cancer is indicated, a therapeutic composition of the invention is preferably administered in a site-directed manner and is repeated as needed. Such therapy may be administered in conjunction with conventional therapies, including radiation and/or chemotherapeutic treatments.

VII. Drug Screening and Development

The proteins, antibodies and polynucleotide sequences of the present invention may also be used in the screening and development of chemical compounds or proteins which have utility as therapeutic drugs for the treatment of cancers characterized by BAP-1 and/or BRCA1 mutation or loss. As one example, a compound capable of binding to BAP-1 and preventing its biological activity may be a useful drug component for the treatment or prevention of cancer. The methods described herein may also be applied to fragments of BAP-1.

Suitable assay methods may be readily determined by one of skill in the art. Where desired, and depending on the assay selected, BAP-1 may be immobilized directly or indirectly (e.g., via an anti-BAP-1 antibody) on a suitable surface, e.g., in an ELISA format. Such immobilization surfaces are well known. For example, a wettable inert bead may be used. Alternatively, BAP-1 may be used in screening assays which do not require immobilization, e.g., in the screening of combinatorial libraries.

Assays and techniques exist for the screening and development of drugs capable of binding to selected regions of BAP-1. These include the use of phage display system for expressing the BAP-1 proteins, and using a culture of transfected E. coli or other microorganism to produce the proteins for binding studies of potential binding compounds. See, for example, the techniques described in G. Cesarini, FEBS Letters, 307(1):66-70 (July 1992); H. Gram et al., J. Immunol. Meth., 161:169-176 (1993); C. Summer et al., Proc. Natl. Acad. Sci., USA, 89:3756-3760 (May 1992), incorporated by reference herein.

Other conventional drug screening techniques may be employed using the proteins, antibodies or polynucleotide sequences of this invention. As one example, a method for identifying compounds which specifically bind to a BAP-1 protein can include simply the steps of contacting a selected BAP-1 protein with a test compound to permit binding of the test compound to BAP-1; and determining the amount of test compound, if any, which is bound to the BAP-1 protein. Such a method may involve the incubation of the test compound and the BAP-1 protein immobilized on a solid support.

Typically, the surface containing the immobilized ligand is permitted to come into contact with a solution containing the BAP-1 protein and binding is measured using an appropriate detection system. Suitable detection systems include the streptavidin horse radish peroxidase conjugate, direct conjugation by a tag, e.g., fluorescein. Other systems are well known to those of skill in the art. This invention is not limited by the detection system used.

Another method of identifying compounds which specifically bind to BAP-1 can include the steps of contacting a BAP-1 protein immobilized on a solid support with both a test compound and the protein sequence which is a receptor for BAP-1 to permit binding of the receptor to the BAP-1 protein; and determining the amount of the receptor which is bound to the BAP-1 protein. The inhibition of binding of the normal protein by the test compound thereby indicates binding of the test compound to the BAP-1 protein.

Thus, through use of such methods, the present invention is anticipated to provide compounds capable of interacting with BAP-1 or portions thereof, and either enhancing or decreasing its biological activity, as desired. Such compounds are believed to be encompassed by this invention.

The assay methods described herein are also useful in screening for inhibition of the interaction between a BAP-1 protein of the invention and its ligand(s). The solution containing the inhibitors may be obtained from any appropriate source, including, for example, extracts of supernatants from culture of bioorganisms, extracts from organisms collected from natural sources, chemical compounds, and mixtures thereof.

These examples illustrate the preferred methods for obtaining and using the sequences and compositions of the invention. These examples are illustrative only and do not limit the scope of the invention.

EXAMPLE 1 Construction of Expression Plasmids

A. LexA Fusions

A totally synthetic BRCA1 gene encoding the amino-terminal 100 amino acids of human BRCA1 (BRCA1-RF), including the full ring-finger domain, was constructed. The BRCA1-RF domain was made using long overlapping oligonucleotides and PCR-mediated overlap-extension gene synthesis techniques [Madden et al, Science, 253:1550-1553 (1991)]. Codon usage was optimized for expression in E. coli and S. cerevisiae [Sharp et al, Nuc. Acids Res., 16:8207-8211 (1988)] (FIGS. 1A and 1B). The following oligonucleotides were used.

top strand: [SEQ ID NO: 10] 5′-ATGGAACCTGTCTGCTCTGCGTGTTGAAGAAGTTCAAAACGTTATCAACGCTATGCAAAAGATCCTGGAATGTCCAATCTG

bottom strand: [SEQ ID NO: 11] 5′-GGTTCAGCAGCTTCAGCATACAGAACTTACAGAAGATGTGGTCACACTTAGTGGAAACTGGTTCCTTGATCAGTTCCAGACAGATTGGACATTCCAGGATC

top strand: [SEQ ID NO: 12] 5′-GTATGCTGAAGCTGCTGAACCAAAAGAAGGGTCCATCTCAATGTCCACTGTGTAAGAACGACATCACTAAGCGTTCTCTGCAAGAATCTACTCGTTTCTCTC

bottom strand: [SEQ ID NO: 13] 5′-TTCCAGACCAGTGTCCAGCTGGAAAGCACAGATGATCTTCAGCAGTTCTTCAACCAGTTGAGAGAAACGAGTAGATTCTTG

Double-stranded DNA was generated by 5 cycles of the polymerase chain reaction (PCR) and the full-length cDNA was amplified further via PCR using “outside” primers with homology to the 5′ and 3′ ends of the DNA sequence [Madden, cited above]. These primers contained enzymatic restriction sites for either EcoRI (BRCA1-RF-5′ primer): 5′-GCTAGAATTCACCATGGACCTGTCTCCTCTG [SEQ ID NO: 14] or Sal I (BRCA1-RF-3′ primer): 5′-GCTAGTCGACTTCCAGACCAGTGTCCAG [SEQ ID NO: 15].

The resulting complete, “wild-type” RF domain was confirmed by sequencing. The resulting BRCA1-RF was then fused in frame with the LexA DNA-binding domain to create a LexA-BRCA1-RF fusion construct by cloning the BRCA1-RF domain into the EcoRI-SalI restriction sites of the vector pBTM-116 [Vojtek et al, Cell, 74:205-214 (1993)] (see FIG. 1B). This LexA-BRCA1-RF construct was used as the probe (“bait) to screen for BRCA1-interacting proteins in a yeast 2-hybrid analysis.

Negative control/specificity controls for the specificity of the interaction in the yeast system were made (as LexA fusions) by mutating the BRCA1-RF (FIGS. 1A and 1B) as follows:

(i) The Cys61Gly and Cys64Gly substitutions of BRCA1 which occur in breast cancer pedigrees. BRCA1 RF domain point mutants, BRCA1-C64G (Cys 64 to Gly) and BRCA1-C61 G (Cys 61 to Gly), were created by PCR-mutagenesis using the “outside” primers described above and overlapping oligonucleotides containing the appropriate nucleotide change: BRCA1-C61 G-sense: 5′-CCATCTCAAGGTCCACTGTGTAAG-3′ [SEQ ID NO: 25]; BRCA1-C61 G-antisense: 5′CTTACACAGTGGACCTTGAGATGG-3′ [SEQ ID NO: 26]; BRCA1-C64G-sense: 5′-CAATGTCCACTGGGTAAGAACGACATC-3′ [SEQ ID NO: 27]; and BRCA1-C64G-antisense: 5′-GATGTCGTTCTTACCCAGTGGACATTG-3′ [SEQ ID NO: 28] [Ho et al, Gene, 77:51-59 (1989)]. The BRCA1(C64G)-RF control has a point mutation in the BRCA1-RF found in a breast cancer kindred [Castilla et al, Nat. Genet., 8:387-391 (1994)]. This mutation, a Cys64 to Gly64, destroys one of the Zn chelating residues leading, presumably, to the loss of correct conformation of the RING domain.

(ii) The protein equivalent of the del AG185 mutation which results in a frame shift at amino acid 22 followed by 17 out-of-frame amino acids and a stop codon. The BRCA1-delAG185 mutant was generated by PCR using the BRCA1-RF-5′ oligonucleotide [SEQ ID NO: 14] and a 3′ oligonucleotide that encoded the changed amino acid sequence: 5′-GCATGGATCCTCAAACCTTGTGCAGGCAGGTACCCTGGTCAACAGGAGACAGGTGGGAAACCAGGATCTTTTGCATAGC-3′ [SEQ ID NO: 29]. The truncated protein generated by the delAG185 mutation is found in high frequency in the Ashkenazi population [Struewing et al, Nat. Genet., 11:198-200 (1995)].

(iii) A truncated BRCA1 RING finger at amino acid 31, the result of a PCR error. The BRCA1-del31 truncation mutant was a mis-primed PCR reaction of BRCA1-RF identified by sequencing during the initial screens for a wild-type LexA-BRCA1. The BRCA1-RF-trunc control is a truncation of the BRCA1-RF, a protein of 35 amino acids which ends within the first loop of the RING domain.

(iv) The RPT-1 RING finger domain. The LexA-RPT-1 construct (amino acids 1-100) [SEQ ID NO: 5] was made by PCR-mediated amplification of the nucleotides representing the first 100 amino acids of the transcription factor RPT-1 [Patarca et al, Proc. Natl. Acad. Sci. USA, 85:2733-2737 (1988); RPT-1 cDNA kindly provided by Dr. H. Cantor] with the 5′ and 3′ primers incorporating EcoRI and Sal I restriction sites.

(v) A non-specific control LexA fusion with RhoB. LexA-RhoB was a kind gift of Dr. George Prendergast, The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.

All LexA mutant fusion constructs were made, as described for the wild-type BRCA1-RF, by cloning the appropriate mutated BRCA1-RF domain into the vector pBTM-116. The RPT-1 PCR product was enzymatically digested and ligated into the corresponding sites in pBTM-116. All clones were confirmed by sequencing.

The wild-type BRCA1-RF did not display intrinsic transcriptional activation function in yeast and proper expression of each LexA fusion in yeast was confirmed by Western blot analysis with anti-LexA DNA-binding domain antibody (data not shown).

These controls screen for proteins which interact only with wild-type the BRCA1-RF and not with any of the physiologically relevant BRCA1-RF mutations nor a RING finger that is the most similar to that of the BRCA1-RF [Miki et al, Science, 266:66-71 (1994)]. Thus, the controls make it possible to identify proteins which interact specifically with the BRCA1-RF and not with any other RING domain.

EXAMPLE 2 Yeast Two-Hybrid Screen for BRCA1-RF Interacting Proteins

To identify the potential protein partners of BRCA1, a yeast 2-hybrid analysis system as modified by Stan Hollenberg [Vojtek et al, cited above] was performed using the RING finger domain of human BRCA1. Guided by the expression patterns of BRCA1 during mouse development and in human spleen, the cDNA libraries selected for screening with the LexA-BRCA1-RF of Example 1 were (1) the human adult B cell, oligo-dT-primed, cDNA library [Durfee et al, Genes & Devel., 7:555-569 (1993) (a kind gift of Dr. Steve Elledge)] and (2) a whole mouse embryo (9.5-10.5 day), random-primed, cDNA library size selected for inserts of 300 to 500 base pairs in length [Vojtek et al, cited above; kind gift of Dr. Stan Hollenberg)].

Briefly, the LexA-BRCA1-RF and a selected library were co-transformed into the L40 yeast strain. Positive protein interactions were selected by His auxotrophy. Fifty colonies were picked and grown for 10 generations without selection for the LexA-BRCA1-RF plasmid. Isolated clones of each colony, which were positive for the presence of only the library plasmid, were picked and mated with AMR70 yeast containing LexA-BRCA1-RF, one of its mutants, or one of the LexA controls of Example 1A. Positive matings were selected by growth on media requiring the presence of both plasmids. These colonies were then scored for LacZ production (positive interaction) and those which were positive for interaction with the wild-type BRCA1-RF, but not any of the controls, were processed for further analysis.

Using the above assay methods, one hundred yeast colonies (50 from each library; each screen representing approximately 8-10×10⁶ independent cDNAs), randomly taken from approximately 5-700 total colonies which grew on solid media lacking the amino acid histidine, were selected for additional screening.

Thirty-one cDNAs which specifically interacted with BRCA1-RF were obtained from the secondary screen of the two libraries. Eight of these (3 from the human library and 5 from the mouse library) encoded the same amino acid sequences.

A representative secondary screen of one of the human clones, hBAP-1 (aa483-729; SEQ ID NO: 6), and 3 of the mouse clones, mBAP-1 (aa581-720; SEQ ID NO: 8), mBAP-1 (aa518-{del}-718; SEQ ID NO: 7), and mBAP-1 (aa596-721; SEQ ID NO: 9) was performed by re-introducing the purified pACT plasmids containing them into naive yeast. The sequences of these clones are compared in FIG. 2B. This screen showed that each clone showed a strong interaction with the wild-type BRCA1 ring-finger, but failed to interact with the C64G, C61G, del3l, delAG, RPT-1, RhoB, or any of the specificity control LexA fusions (data not shown).

Thus, these clones specifically interact with only the BRCA1 RING finger. These cDNA clones all encode the same region of the same protein which has been termed BRCA1-Associated Protein-1 or BAP-1. Each clone shares the same translational reading frame with the transcriptional activation domain to which it is fused. In addition, the fusion junctions were different among the clones suggesting that the interaction was not due to a fusion-junction artifact.

The longest cDNA retrieved in the two-hybrid screen was a 2.0 kb clone from the human library and encoded 246 amino acids followed by a 1.3 kb 3′UTR. Each mouse clone encoded an overlapping, smaller subset of this human open reading frame and which served to partially map the minimal interaction domain. Further definition of this minimal interaction domain was performed by mutagenesis of this region of BAP1.

The “minimal interaction domain” was determined by the shortest mouse clone [mBAP-1 (aa596-721; SEQ ID NO: 9)]. To further define the specificity of interaction between BRCA1 and BAP-1, carboxy- and amino-terminal truncation mutants of mBAP-1 were generated by PCR-based deletion or point mutagenesis.

The appropriate region of mBAP1(596-721) was amplified by PCR using a vector primer pVP16 5′-primer, 5′-CCGATGCCCTTGGAATTGACGAG-3′; pVP16 3′-primer, 5′-CGATGAATTCGAGCTAGCTTCTATC-3′ ) and the appropriate truncating oligonucleotide Mc43Ct1, 5′-GCATGAATTCTCAGCT CCGGCGCACTGAGATG-3′; Mc43Ct2, 5′-GCATGAATTCTCAAGCCAGCATGGATATGAAGG-3′; Mc43Ct3, 5′-GCATGAATTCTCAGTCATCAATCTTGAACTTC-3′; Mc43Ct4, 5′-GCATGAATTCTCATGCAATCTCGGCTTCTAC-3′; or Mc43Nt1, 5′-GCATG GATCCCCAAGATTGATGACCAGCGAAGG-3′ [SEQ ID NOS: 30 to 36, respectively).

These oligonucleotides were generated with an incorporated EcoRI restriction site (for the 3′ end oligos) or a BamHI restriction site (for the 5′ end oligos). After PCR amplification, the product was cut with BamHI and EcoRI, and then ligated into the mouse library-yeast expression vector, pVP16 [Vojtek et al, cited above].

The point mutant mBAP-1 (L691P) was made by standard PCR-based mutagenesis protocols [Ho et al, Gene, 77:51-59 (1989)], using (Mc43(L691 P) sense-primer, 5′-GCTGGCCAACCCGGTGGAACAG-3′ [SEQ ID NO: 37]; Mc43(L691P) antisense-primer, 5′-CTGTTCCACCGGGTTGGCCAGC-3′ [SEQ ID NO: 38] and using the same vector primers described above.

The “minimal interaction domain” was deleted from the human sequence (the longest clone) and this protein (hBAP-1(483-594) [SEQ ID NO: 6] was also assayed for interaction with the BRCA1-RF in the yeast 2-hybrid system.

All clones were confirmed by sequencing and expression in yeast was confirmed by western analysis using antibodies against the VP16 activation domain (data not shown). Each individual mutant was co-transformed with LexA-BRCA1-RF into L40 yeast and tested for interaction via its ability to activate transcription from the LacZ locus.

The mutants showed that deletion of protein sequence from the carboxy or amino termini of mBAP-1 (aa 596-721; SEQ ID NO: 9) almost completely destroyed the BAP1-BRCA1 interaction, suggesting a complex interface between the proteins. Deletion of the last 20 amino acids of mBAP-1 led to a significant reduction in the intensity of interaction. Further deletions from the COOH-terminus led to the complete loss of interaction between BRCA1-RF and BAP-1. A single amino-terminal truncation which deleted approximately half of mBAP-1 (aa 596-721; SEQ ID NO: 9) led to an almost complete loss of interaction. Interestingly, the mBAP(518del718) clone interacted most poorly with BRCA1-RF and lacked a 93 bp sequence (the reading frame was maintained), possibly the result of a naturally occurring splice variant. That these clones also fail to bind multiple, independent tumor-derived mutations of the BRCA1-RF provides strong genetic evidence for their relevance to the functions of BRCA1.

The results of the above experiments suggested that some critical domain was being disrupted by these truncations. A careful analysis showed that the region from amino acids 632 to 729 of SEQ ID NO: 6 may in fact generate a coiled-coil domain. A point mutation in the middle of the domain (leucine 691 substituted with a proline) destroys interaction with the BRCA1 RING structure. This result is consistent with the BAP-1/BRCA-1 interaction domain being a coiled-coil.

EXAMPLE 3 Analysis of BAP-1 cDNA

A nearly full-length cDNA was constructed via a combination of cDNA library screening, EST database searching, 5′RACE and RT-PCR (FIGS. 3A-3E) as follows. Searches of the protein and DNA databases [Altschul et al, J. Mol. Biol., 215:403-410 (1990)] with the BAP-1 protein/cDNA sequences obtained from the screening of Example 2, showed no significant matches with any known protein or cDNA. However, searches of the EST databases with BAP-1 cDNA yielded several “hits”, including one whose clone had a 5′ sequence that overlapped with the 3′ sequence of another EST clone. The clones defined by these EST's were obtained from the I.M.A.G.E. consortium [Lennon et al, Genomics, 33:151-152 (1996); clones #46154 and #40642]. A partial BAP-1 cDNA clone (EST-BAP1) was generated by digesting clone #40642 with Hind III and Fsp I and clone #46154 with Fsp I and EcoRI. These two pieces were then ligated into the Hind III and EcoRI sites of the vector pcDNA3 (Invitrogen).

Analysis of the IMAGE consortium cDNA and its open-reading-frames suggested that this BAP-1 cDNA, as constructed, was not complete. Reverse-transcriptase-PCR was performed on RNA from normal human fibroblasts using a gene-specific primer: 5′-GAAGCGGATGTCGTGGTAGG-3′ [SEQ ID NO: 43] and identified 62 nucleotides which were missing from the “EST-BAP1” cDNA. These 62 nucleotides were inserted into the “EST-BAP1” cDNA by digestion of the RT-PCR product with the restriction enzymes KpnI, which is a unique site within the 5′ RT-PCR oligonucleotide: 5′CCTGTTATTAACCCTCACTAAAGGGAAGGGTACCATGAATAAGGGCTGGCTGGAGC-3′ [SEQ ID NO: 39] and 3′ RT-PCR-oligonucleotide: 5′-GAAGCGGATGTCGTGGTAGG-3′ [SEQ ID NO: 40, and AvrII. Ligation of the KpnI/AvrII digested RT-PCR fragment and AvrII/EcoRI digested “EST-BAP1” cDNA and the KpnI-EcoRI digested pcDNA3, produced the full-length BAP-1 cDNA.

Thus, BAP1 cDNA (FIGS. 3A to 3E; SEQ ID NO: 1] comprises 3525 bp, including a polyA tract with multiple polyA signals. Conceptual translation yields a long open reading frame of 729 amino acids [SEQ ID NO: 2] with a predicted MW of about 81 kDa and pI of 6.3.

The presumptive initiator methionine is within a favorable context for translation start, however the short 5′UTR of 39 bp encodes amino acids in-frame with the presumptive methionine and does not contain a stop codon. BLAST searches and a domain analysis [Henikoff & Henikoff, Genomics, 19:97-107 (1994)] indicated that BAP1 is a novel protein with motifs suggestive of function.

The amino-terminal 1-240 amino acids of SEQ ID NO: 2 show significant homology to a class of thiol proteases, designated ubiquitin C-terminal hydrolase (UCH), particularly Isozyme L3, which are implicated in the proteolytic processing of ubiquitin [Wilkinson et al, Science, 246:670-673 (1989)]. These enzymes play a key role in protein degradation via the ubiquitin-dependent proteasome pathway. The most closely related UCH is a hypothesized protein from C. elegans UCH-CAEEL, which shares 63% similarity (40% identity) with BAP1 through the UCH domain and is also likely to be a UCH enzyme. Pairwise similarities to other mammalian UCHs of 54% (UCHL3) and 56% (UCHL1) have also been found. Most importantly, the residues which form the catalytic site of BAP1 (Q85, C91, H169, and D184 of FIGS. 3A-3E; SEQ ID NO: 2) are completely conserved, including the FELDG motif [Larsen et al, Biochemistry, 35:6735-6744 (1996)]. In addition, a loop of highly variable sequence, which is disordered in the crystallographic structure of human UCH-L3 [Johnston et al, EMBO J., 16:3787-3796 (1997)], is present (residues 140 to 167 of SEQ ID NO. 2). This loop may occlude the active site or provide substrate specificity for the enzyme.

BAP1 has a number of additional motifs; a region of extreme acidity spanning amino acids 396 to 408 of SEQ ID NO. 2, as well as multiple potential phosphorylation sites and N-linked glycosylation sites. The C-terminal one-third is highly charged and is rich in proline, serine and threonine. The extreme C-terminus contains two putative nuclear localization signals, KRKKFK and RRKRSR (aa 656-661 and aa 717-722 of SEQ ID NO: 2), and is hydrophilic; it is predicted to fold into a helical (possibly coiled-coil) structure. Indeed, within the BAP1 minimal interaction domain, (i.e., from about amino acid 596 to 729 of SEQ ID NO: 2) the mutation of leucine 691 to a proline, a change predicted to disrupt the helical nature of this region, abolished the BAP1-BRCA1 interaction, consistent with the hypothesis that BAP1 uses a coiled-coil domain to interact with the RING finger domain of BRCA1. This overall architecture suggests that BAP1 is a new, structurally complex, and nuclear localized member of the UCH enzyme family.

EXAMPLE 4 In Vitro Protein Association

The direct interaction of the BRCA1-RF with BAP-1 was confirmed by binding of the BRCA1-RF to the fusion proteins of glutathione-S-transferase with BAP1.

A. The BAP/GST Constructs

The original B cell library two-hybrid BAP-1 clone obtained from the screening experiments described in Example 2 was pACT-hBAP1(483-729), which contained BAP1 amino acids 483-729 (nucleotides 1486 to 3525) [SEQ ID NOs: 2 and 1, respectively)] in the pACT plasmid backbone. The glutathione S-transferase/BAP1 fusion protein, GST-hBAP1(483-729 of SEQ ID NO: 2), was generated by cloning nucleotides 1486 to 3525 of SEQ ID NO: 1 from that original clone into pGEX-5x-1 (Pharmacia Biotech, Inc.).

Another BAP1 construct which lacked the minimal BRCA1 interaction domain pACT-hBAP1(483-594 of SEQ ID NO: 2), was generated and amplified by PCR using a pACT 5′ vector primer 5′-GATGTATATAACTATCTATTCG-3′ [SEQ ID NO: 41] and the BAP 1-trunc. oligonucleotide: 5′-GCATAGATCTTCACCCCTGGCTGCCTTGGATTGG3′ [SEQ ID NO: 42], which amplifies BAP1 nucleotides 1486-1821 of SEQ ID NO: 1. The resulting sequence was digested with restriction enzymes and ligated into the vector pACT. Another fusion protein GST-hBAP1(483-594 of SEQ ID NO: 2) lacking the minimal BAP1 interaction domain, was generated in the same manner as pACT-hBAP1(483-594 of SEQ ID NO: 2), described above, but fused to GST.

GST, and the BAP1 fusion constructs GST-hBAP1(483-729 of SEQ ID NO: 2) and GST-hBAP1(483-594 of SEQ ID NO: 2), were expressed in E. coli and then purified [Frangioni et al, Anal. Biochem., 210:179-187 (1993)]. ³⁵S-LexA-BRCA1-RF and ³⁵S-BRCA1 were produced in vitro via coupled transcription/translation (TNT®, Promega Corp., Madison, Wis.) in the presence of ³⁵S-Met.

B. Association Assay

Association between the proteins was assayed essentially as described by Barlev et al, J. Biol. Chem., 270:19337-19344 (1995). Briefly, each GST resin was incubated with the LexA-BRCA1-RF in 100 μL of incubation buffer (PBS containing 0.2 mM ZnSO₄, 0.05% NP-40 and 1 mM PMSF) for 1 hour at 4° C. followed by a second hour at room temperature. The resin and associated proteins were then washed in incubation buffer twice (1 mL at room temperature for 15 minutes) followed by four washes in PBS containing 300 mM NaCl, 0.2 mM ZnSO₄, 0.1% NP-40 and 1 mM PMSF. The associated proteins which remained bound to resin were eluted from the resin two times (15 minutes), each with 250 μL of elution buffer (100 mM TRIS, pH 8.0, 150 mM NaCl, 0.1% NP-40, 20 mM reduced glutathione). The two elutions were combined, concentrated to a volume of approximately 20 μL of a 50:50 resin slurry, and analyzed by SDS-PAGE and visualized by Coomassie blue staining and fluorography.

Association of the BRCA1-RF with BAP1 was confirmed in vitro by specific binding of ³⁵S-labeled LexA-BRCA1-RF to GST-hBAP1(483-729 of SEQ ID NO: 2) fusion protein, but not to GST alone, confirming a physical association of the two proteins.

To confirm that the association of the BRCA1-RF to BAP1 was not an artifact of using only a portion of BRCA1, full length BRCA1 was expressed in vitro and incubated with GST and GST-hBAP1(483-729 of SEQ ID NO: 2). As a further control for the specificity of the interaction, BRCA1 was also incubated with GST-hBAP1(483-594 of SEQ ID NO: 2), the GST-BAP1 fusion protein lacking the minimal interaction domain.

The BRCA1 protein specifically bound to GST-hBAP1(483-729 of SEQ ID NO: 2) and not to GST or GST-hBAP1(483-594 of SEQ ID NO: 2), confirming the direct interaction of BRCA1 with BAP1 through the C-terminal region of BAP1.

EXAMPLE 5 Generation of Antibodies

Oligonucleotide primers (pACT 5′-vector primer 5′-GATGTATATAACTATCTATTCG-3′ [SEQ ID NO: 44′; BAP1 3′ primer (antibody) 5-CGTAGTCGACTGTCAGCGCCAGGGGACTC-3′ [SEQ ID NO: 45]), were used to amplify the portion of the BAP1 cDNA [SEQ ID NO: 1] corresponding to amino acids 483 to 576 of SEQ ID NO: 2 via PCR cloning. The PCR product was then digested with the appropriate restriction enzymes and ligated to the COOH-terminus of 6 Histidine residues of the vector pQE-30 (QIAGEN Inc.).

The His-tagged protein was purified from E. coli over a Ni-agarose column as described [Friedman et al, cited above] and was used to immunize rabbits for the production of polyclonal antibodies (Cocalico Biologicals, Inc.).

EXAMPLE 6 Protein Expression of BAP1

COS-1 cells were grown at 37° C., 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 2 mM L-glutamine. COS1 cells were transiently transfected using DOSPOR transfection reagent (Boehringer Mannheim Biochemicals) following the manufacturers protocol with plasmids containing the BAP1 cDNA, e.g., pACT-hBAP1(483-729 of SEQ ID NO: 2). The BAP1 cDNA was transcribed and translated in vitro in the presence of ³⁵S-Methionine. ³⁵S-labeled cytosolic and nuclear extracts were then prepared from transiently transfected COS1 cells.

Immunoprecipitation of BAP1 was performed by previously described procedures for the metabolic labeling and immunoprecipitation of proteins from cell lysates [Morris et al, Oncogene, 6:2339-2348, (1991); Rauscher et al, Science, 240:1010-1016 (1988); Friedman et al, cited above] with either pre-immune or anti-BAP1 seras described in the above example.

As a control for nuclear localization, KAP-1, a co-repressor of transcription localized to the nucleus (Friedman et al, Genes Dev., 10:2067-2078, (1996)], was also immunoprecipitated from these cell fractions. Immunoprecipitation of this product with anti-BAP-1 antiserum confirmed that the protein expressed in vitro from the cDNA resulted in a polypeptide that contained the antigen used to raise the antibodies produced as described above. BAP-1 was found primarily in the nuclear fraction although a significant amount was detected in the cytosol. However, this may be an artifact of the cell fractionation procedure, since KAP-1 was also found to be present in both cytosolic and nuclear fractions and in approximately the same ratio as BAP-1.

The expression of the BAP-1 cDNA in COS1 cells in vitro followed by immunoprecipitation of ³⁵S-labeled whole cell extract and analysis by SDS-PAGE also yielded a single major protein with an apparent molecular weight of about 91 kDa. However, the largest BAP1 open reading frame encodes a protein of about 81 kDa predicted molecular weight. The difference between apparent and predicted molecular weights may be accounted for by unusual properties of the C-terminus or by post-translational modifications.

EXAMPLE 7 Tissue and Cellular Expression of BAP-1

A. BAP1 is Expressed in a Variety of Tissues

The direct interaction between BAP1 and BRCA1 illustrated in Example 4, suggests that BAP1 might be expressed in an overlapping subset of tissues expressing BRCA1 and that the subcellular location of BAP1 and BRCA1 may be the same.

The expression of BAP1 in a variety of human adult tissues was determined by Northern blot analysis. Northern blot hybridizations were performed as follows: Ten μg total RNA from multiple tissue RNA blots (Clontech Laboratories, Inc., Palo Alto, Calif.), was electrophoretically gel-fractionated and transferred to Hybond N+ membranes (Amersham). The tissues represented were heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood lymphocytes.

The protocols for hybridization of cDNA probes to RNA were performed as described (Clontech Laboratories, publication PR48380). Blots were hybridized with a 2.0 kbp ³²P-1abeled hBAP1 cDNA (aa483-729; nucleotides 1486 to 3525) followed by washes under standard conditions and detection by autoradiography. Blots were also subsequently probed with a muscle actin cDNA.

The results indicated that the mRNA encoding BAP1 was present as a single mRNA species of about 4 kb in all tissues except testis, where a second, about 4.8 kb mRNA, was also detected. Highest expression was detected in testis, placenta and pancreas with varying levels detected in the remaining tissues. Expression of BAP1 in normal breast tissue was confirmed by RT-PCR of total RNA isolated from normal human mammary epithelial cells (HUMEC; data not shown). The level and pattern of tissue expression shown by BAP1 is similar to that shown by BRCA1 [Miki et al, cited above].

Northern blot analysis was also performed on several tumor cell lines representing a variety of tissue types. The cell line RNA blot was prepared by standard methods (Sambrook et al, cited above) with 20 μg of total RNA. Equivalent loading of RNA was confirmed by ethidium bromide staining. Hybridization of cDNA probes to RNA were performed using the Clontech protocols. This hybridization also showed a single mRNA species. The colon cell lines HT29 [ATCC HTB 28] and SK-Co-1 [ATCC HTB 39] showed no BAP-1 mRNA, suggesting some defect in the BAP-1 gene in these particular cell lines since colon tissue shows good expression of BAP-1.

B. BAP1 is a Nuclear Protein

The location of BAP1 as a nuclear protein within the cell was determined by immunofluorescence microscopy performed as previously described [Ishov et al, J. Cell Biology, 134:815-826 (1996)]. HEP2 epithelial cells were grown at 37° C., 5% CO₂ in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, and cells were transfected using DOSPOR transfection reagent (Boehringer Mannheim Biochemicals) following the manufacturers protocol via electroporation with the pcDNA3 vector (Invitrogen, Inc.) carrying the BAP1 cDNA.

Transfectants were analyzed by immunofluorescence staining with anti-BAP1 polyclonal antibodies, which in turn, were detected with FITC using biotin-avidin enhancement. Cells were stained for DNA with bis-benzimide (Hoechst 33258, Sigma Chemical Co.) and mounted using Fluoromount G (Fisher Scientific). Analysis was performed with a confocal scanning microscope (Leica, Inc.).

Detection of BAP1 by confocal microscopy located BAP1 almost exclusively in the nucleus of the cell consistent with its association with BRCA1, and the presence of two nuclear localization signals in the BAP1 protein sequence.

C. BAP1 is Located on Chromosome 3p21.3 and is Mutated in Non-Small Cell Lung Carcinoma.

To determine whether BAP1 was located at a chromosomal region routinely mutated in breast cancer and thus may be a tumor suppressor gene, the deletion of which plays a critical role in tumor pathogenesis, full-length BAP-1 cDNA was used in fluorescent in situ hybridization (FISH) of partial metaphases. FISH was performed as described previously [Tommerup and Vissing, Genomics, 27:259-264 (1995)] using a biotin-labelled 3.5 kb cDNA (full-length) clone of BAP-1, with corresponding DAPI-stained chromosome banding. Localization of BAP1 was based on the DAPI-band pattern and measurement of the relative distance from the short arm telomere to the signals (FLpter value).

BAP1 maps to chromosome 3p21.3. Specific signals were observed only on the midportion of the short arm of chromosome 3 with 42 of 69 analyzed metaphase spreads showing at least one specific signal. The FLpter value was 0.27+0.02, corresponding to a localization for BAP1 at 3p21.2-p21.31. This location is a region of LOH for breast cancer as well as a region frequently deleted in lung carcinomas [Buchhagen et al, Int. J. Cancer, 57:473-479 (1994); Thiberville et al, Int. J. Cancer, 64:371-377 (1995)].

EXAMPLE 8 Mutational Analysis of BAP1

The chromosomal location of BAP1 suggested the possibility of mutations within BAP1 in lung and breast tumors. Thus, a variety of tumor cell lines were screened for mutations within the BAP1 gene by Southern, Northern and PCR-based SSCP analyses.

A. RNA/DNA Preparation

Genomic DNA from a panel of small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), breast cancer, and lymphoblastoid cell lines was prepared using standard methods. All cell lines were identified by their NCI number [Phelps et al, J. Cell. Biochem. Suppl., 24:32-91 (1996)]: H727, H1466, H226, H526, H841, H1045, H289, BL1672, BL1770, H289, H847, H920, H1450, H1573, H1155, H1299, H1693. Total RNA was extracted by the cesium chloride ultracentrifugation method [Ausubel et al, Current Protocols in Molecular Biology, J. Kaaren ed., John Wiley & Sons, Inc. (1987)]. First strand cDNAs were synthesized from RNA by M-MLV reverse transcriptase (Gibco BRL) according to the manufacturer's instructions.

B. Single Strand Conformational Polymorphism (SSCP) Analysis

Seventeen overlapping PCR primer pairs, each with a predicted product size of approximately 200 base pairs, were designed to span the 2.2 kb open reading frame of the BAP1 cDNA sequence. cDNA (from RNA) was amplified in 20 μl PCR reactions containing 20 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 0.2 mM each dNTP, 0.1 mM each forward and reverse primer, 0.05 ml 32P-α dCTP, and 0.5 units Taq DNA Polymerase (BRL). PCR reactions were carried out in a Perkin-Elmer 9600 Thermocycler using a touchdown technique: a 2.5 minute initial denaturation at 94° C. was followed by 35 cycles of denaturation at 94° C.×30s, annealing, initially at 65° C. decreasing by 1° C. for each of the first ten cycles to 55° C.,×30s, and extension at 72° C.×30s with a final extension of 5 minutes at 72° C. PCR products were then diluted 1:10 with SSCP dye (95% formamide, 20 mM EDTA, and 0.05% each of bromophenol blue and xylene cyanol), heat denatured, and electrophoresed on 0.5×MDE gels +/− 10% glycerol. Abnormal single stranded DNA detected as autoradiographic shifts were reamplified by PCR and subjected to automated dye-terminator sequencing (ABI 373).

SSCP analysis showed a homozygous shift in H1466 detected by RT-PCR amplification spanning nts 1089 to 1286 (primers: sense 5′-CAACCCCACTCCCATTGTC-3″ [SEQ ID NO: 46]; antisense 5′-GAGTTGGTGTTCTGCACGTC-3″ [SEQ ID NO: 47]). Automated sequencing revealed a homozygous 8 base pair frameshift deletion in the NCI-H1466 cDNA, predicted to encode a truncated 393 amino acid BAP1 protein. This homozygous deletion was confirmed to be present in genomic DNA from the same cell line. In the NCI-H226 line, only the 2.4 kb band and an aberrant 2.6 kb band were detected.

B. Northern Analysis

These cell lines were subjected to Northern blot analysis and EcoRI digestion and then hybridized to a full-length BAP1 cDNA probe. A single 23 kb band was detected in the lymphoblastoid and most tumor cell lines (data not shown). One NSCLC line, NCI-H226, did not show the 23 kb band but did show an aberrant 30 kb band (data not shown).

Further mutational analysis was performed by screening a panel of lung cancer and lymphoblastoid cell lines for expression of BAP1 mRNA. Northern blot hybridization showed that most cell lines expressed a single 4 kb mRNA. A fainter (5.0 kb) band was visible corresponding to cross-hybridization with the 28S ribosomal component. However, two cell lines, NCI-H226 and the non-small cell lung cancer NCI-H1466 (both NSCLCs), showed undetectable levels of BAP1 expression, suggesting that BAP1 may play a critical role in NSCLC pathogenesis.

C. Southern Analysis

To further characterize this potential genomic rearrangement, genomic DNA from NCI-H226 and a smaller number of lung cancer and lymphoblastoid lines were subjected to Southern blot hybridization. Briefly, five μg of genomic DNA was subjected to restriction enzyme digestion with BamHI. Using the full-length BAP1 cDNA probe, four distinct bands at 7.5 kb, 4.0 kb, 3.0 kb, and 2.4 kb were detected which were present in all cell lines tested with the exception of NCI-H226. The non-small cell lung cancer NCI-H226 line shows an absence of the 7.5 kb, 4.0 kb, and 3.0 kb bands. An aberrant 2.6 kb band is detected in the NCI-H226 cell line.

These data clearly show that genetic alterations, including intragenic homozygous deletions, occur in BAP1.

EXAMPLE 9

BAP1 Augments the Growth Suppressive Activity of BRCA1

To determine whether BAP1 may affect cell growth itself or may affect BRCA1-mediated changes in cell growth, BRCA1 and BAP1 cDNAs were co-transfected into MCF7 breast cancer cells. This cell line was chosen for several reasons. It has been previously shown that these cells are inhibited by the overexpression of BRCA1 [Holt et al, cited above]. Both northern and RT/PCR analyses showed that BAP1 was expressed in this cell line (data not shown); and analysis of the open reading frame from BAP1 cDNA prepared from this cell line showed no mutations (data not shown).

MCF7 cells grown at 37° C., 5% CO2 in DMEM supplemented with 10% FBS and non-essential amino acids, were transfected with the following plasmid pairs: (a) empty plasmids pcDNA3 and pCMV5; (b) pcDNA3 and pCMV5-BAP1; (c) pcDNA3 and pCMV5-BAP1(165-729 of SEQ ID NO: 2); (d) pcDNA3-BRCA1 and pCMV5; (e) pcDNA3-BRCA1 and pCMV5-BAP1; (f) pcDNA3-BRCA1 and pCMV5-BAP1(165-729 of SEQ ID NO: 2); (g) pcDNA3-BRCA1-Δ11 and pCMV5; (h) pcDNA3-BRCA1-Δ11 and pcMV5-BAP1; and (i) pcDNA3-BRCA1-Δ11 and pCMV5-BAP1(165-729 of SEQ ID NO: 2) by a modified CaPO₄-DNA precipitation method [Holt et al, cited above].

MCF7 cells, at 2×10⁶ cells/10 cm dish, were fed fresh medium approximately 3 hours prior to transfection and were then treated with the Ca-DNA precipitate for 4 hours. The cells were subjected to a brief shock with transfection buffer containing 15% glycerol. Twelve to sixteen hours later, the cells were trypsinized, counted and plated directly into complete medium containing 0.75 mg/mL G418 at 5×10⁵ cells per 10 cm dish. Cells were fed fresh medium containing G418 every three to four days. Cells were stained for colonies approximately 21 to 28 days after transfection. The experiment was repeated 4 times with similar results.

The expression of BRCA1 alone (pcDNA3-BRCA1:pCMV5) decreased the number of colonies formed by these cells when compared to the empty vector control (pcDNA3:pCMV5), in agreement with other studies [Holt et al, cited above]. The co-expression of BRCA1 and BAP1 (pcDNA3-BRCA1:pCMV5-BAP1) significantly decreased the number of cell colonies (approximately 4 fold vs. BRCA1 alone) indicating that BAP1 enhances the growth suppressive actions of BRCA1. A mutant of BAP1, BAP1(AA165-729), in which the enzymatic region is deleted but which still binds to BRCA1 (data not shown), also enhanced the growth suppression of BRCA1, but not to the same extent as the wildtype BAP1.

In contrast to BRCA1, the expression of BRCA1-Δ11 (BRCA1 missing the 11th exon) in MCF7 cells by itself had no effect on the growth of MCF7 cells. However, the co-expression of BRCA1-Δ11 and BAP1 significantly decreased the number of colonies, suggesting that the presence of BAP1 could functionally substitute for the missing 11th exon of BRCA1 and/or that BAP1 itself was an inhibitor of cell growth.

In support of this latter hypothesis, the expression of BAP1 in MCF7 cells did somewhat reduce the number of colonies formed (pcDNA3:pCMV5-BAP1). The expression of the enzymatic mutant, BAP1(165-729), alone or in combination with BRCA1-Δ11 yielded the same number of colonies. Thus, enzymatically active BAP1 enhances BRCA1-mediated suppression of growth.

EXAMPLE 10 BAP1 Enzymatic Assay

To determine whether BAP1 did indeed have UCH activity, the BAP1 cDNA was expressed in bacteria and this protein was assayed for the ability to hydrolyze the glycine 76 ethyl ester of ubiquitin [Ub-OEt; Mayer et al, Biochemistry, 28:166-172 (1989)].

Briefly, bacteria (E. coli DH5a) harboring an IPTG-inducible expression plasmid containing BAP1 or an enzymatically null mutant, BAP1 (C91 S) (pQE-30; QIAGEN Inc.) were grown and induced with 1 mM IPTG for 4 hours. The bacteria were collected and the pellets were resuspended to {fraction (1/20)} volume (original culture) in lysate buffer (50 mM Tris, pH 8.0, 25 mM EDTA, 10 mM 2-mercapto-ethanol, 100 μg/ml lysozyme). The lysates were sonicated and centrifuged at 40,000×g.

The pellets were resuspended in an volume equal to that of the supernatant and samples of both pellet and supernatant were analyzed by SDS-PAGE for expression levels and inclusion body formation. Induction of protein was verified by SDS-PAGE of each fraction. Overexpression of BAP1 in bacteria led to abundant protein, most of which was found in an inactive, insoluble form.

Assays for BAP1 enzymatic activity, specifically, ubiquitin carboxy-terminal hydrolase activity, were performed on the above-described soluble fraction essentially as described for the UCH-L1 and UCH-L3 enzymes using the glycine 76 ethyl ester of ubiguitin (Ub-OEt) as a substrate [Mayer et al, cited above; Wilkinson et al, Biochemistry, 25:6644-6649 (1986)]. Assays were done in triplicate. The peak areas were integrated and normalized with respect to a ubiquitin standard.

The BAP1 protein found in the soluble fraction was able to hydrolyze UbOEt and the level of this activity increased with the level of protein, indicating that BAP1 contains UCH-like enzymatic activity.

The active site thiol residue responsible for UCH activity in UCH-L3 has been identified and its mutation leads to abolition of enzyme activity [Larsen et al, cited above]. Mutation of the corresponding cysteine residue in BAP1, BAP1 (C91 S), yielded a protein with no UCH activity, further suggesting that BAP1 is a thiol protease of the UCH family.

BAP1's identity as a protease of the ubiquitin carboxy-terminal hydrolase (UCH) family implies a role for either ubiguitin-mediated, proteasome dependent degradation or other ubiquitin-mediated regulatory [Isaksson et al, Biochimica et Biophysica Acta, 1288:F21-29 (1996)] pathways in BRCA1 function. Regulated ubiquitination of proteins and subsequent proteasome-dependent proteolysis plays a role in almost every cellular growth, differentiation and homeostatic process (reviewed by Ciechanover, Biol. Chem. Hoppe-Seyler, 375:565-581 (1994); Isaksson et al, cited above; Wilkinson, Annual Review of Nutrition, 15:161-189 (1995)]. This pathway can be broadly subdivided into reactions involving 1) pro-ubiquitin processing and ATP-dependent activation of ubiquiting; 2) substrate recognition, conjugation and editing of the polyubiquitin chain; 3) proteasome-dependent degradation of the ubiquitin protein and; 4) cleavage and/or debranching of peptide-ubiquitin conjugates and recycling of ubiquitin to cellular pools. The pathway is regulated at almost every step. First, at the level of substrate specificity via the concerted actions of activating enzymes, carrier proteins and ligation enzymes, and secondly, at the level of proteolytic deubiquitination and ubiquitin hydrolysis.

The UCH family has been characterized as a set of small (25-30 kDa) cytoplasmic proteins which prefer to cleave ubiquitin from ubiquitin-conjugated small substrates and may also be involved in the co-translational processing of proubiquitin. UCHs show considerable tissue specificity and developmentally-timed regulation [Wilkinson et al, Biochem. Soc. Trans., 20:631-637 (1992)]. UCH family members are strongly and differentially expressed in neuronal, hematopoietic and germ cells in many species. Most remarkably, a novel UCH enzyme has recently been cloned from Aplysia californica whose enzymatic function is essential for acquisition and maintenance of long-term memory [Hedge et al, Cell, 89:114-126 (1997)]. Finally, UCH levels are strongly downregulated during viral transformation of fibroblasts [Honore et al, FEBS Letter, 280:235-240 (1991)], consistent with a role in growth control.

BAP1 is the newest member of the UCH family and considerably expands the potential roles of this family of proteases. BAP1 is a much larger protein (90 kDa) and is the first nuclear-localized UCH. BAP1 is also likely to be involved in the regulation of protein subcellular localization. Ubiquitin, or a ubiquitin-like moiety, may affect the specific targeting of proteins to locations other than the proteasome [see, e.g., Mahajan et al, Cell, 88:97-107 (1997)]. BAP1-mediated removal of “ubiquitin” from BRCA1, or a protein associated with BRCA1, could target it for removal to another cellular compartment, thus functionally destroying the protein without physically doing so.

BRCA1 is also localized in nuclear dot structures in a cell-cycle dependent manner [Scully et al, cited above]. This association of BRCA1 with RAD51 in both mitotic and meiotic cells broadly implicates BRCA1 in DNA repair and/or recombination processes. The RAD51/52-dependent DNA repair pathway is highly regulated and includes many proteins, some of which may be potential substrates for BAP1-mediated ubiquitin hydrolysis [Watkins et al, Molecular & Cellular Biology, 13:7757-7765 (1993)]. Thus, it appears that the DNA repair machinery contains both ubiquitin-conjugating and -hydrolyzing elements, since BAP1 is now implicated as a member of the BRCA1/RAD51/hUBC9 complex. It is possible that BAP1, which is co-expressed with BRCA1 in testis, may regulate the recombination/repair functions of the BRCA1/RAD52 complex by targeting either RAD23 or UBL1 for ubiquitin hydrolysis.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

47 3517 base pairs nucleic acid double Not Relevant cDNA not provided CDS 40..2226 1 GGCACGAGGC ATGGCGCTGA GGGGCCGCCC CGCGGGAAG ATG AAT AAG GGC TGG 54 Met Asn Lys Gly Trp 1 5 CTG GAG CTG GAG AGC GAC CCA GGC CTC TTC ACC CTG CTC GTG GAA GAT 102 Leu Glu Leu Glu Ser Asp Pro Gly Leu Phe Thr Leu Leu Val Glu Asp 10 15 20 TTC GGT GTC AAG GGG GTG CAA GTG GAG GAG ATC TAC GAC CTT CAG AGC 150 Phe Gly Val Lys Gly Val Gln Val Glu Glu Ile Tyr Asp Leu Gln Ser 25 30 35 AAA TGT CAG GGC CCT GTA TAT GGA TTT ATC TTC CTG TTC AAA TGG ATC 198 Lys Cys Gln Gly Pro Val Tyr Gly Phe Ile Phe Leu Phe Lys Trp Ile 40 45 50 GAA GAG CGC CGG TCC CGG CGA AAG GTC TCT ACC TTG GTG GAT GAT ACG 246 Glu Glu Arg Arg Ser Arg Arg Lys Val Ser Thr Leu Val Asp Asp Thr 55 60 65 TCC GTG ATT GAT GAT GAT ATT GTG AAT AAC ATG TTC TTT GCC CAC CAG 294 Ser Val Ile Asp Asp Asp Ile Val Asn Asn Met Phe Phe Ala His Gln 70 75 80 85 CTG ATA CCC AAC TCT TGT GCA ACT CAT GCC TTG CTG AGC GTG CTC CTG 342 Leu Ile Pro Asn Ser Cys Ala Thr His Ala Leu Leu Ser Val Leu Leu 90 95 100 AAC TGC AGC AGC GTG GAC CTG GGA CCC ACC CTG AGT CGC ATG AAG GAC 390 Asn Cys Ser Ser Val Asp Leu Gly Pro Thr Leu Ser Arg Met Lys Asp 105 110 115 TTC ACC AAG GGT TTC AGC CCT GAG AGC AAA GGA TAT GCG ATT GGC AAT 438 Phe Thr Lys Gly Phe Ser Pro Glu Ser Lys Gly Tyr Ala Ile Gly Asn 120 125 130 GCC CCG GAG TTG GCC AAG GCC CAT AAT AGC CAT GCC AGG CCC GAG CCA 486 Ala Pro Glu Leu Ala Lys Ala His Asn Ser His Ala Arg Pro Glu Pro 135 140 145 CGC CAC CTC CCT GAG AAG CAG AAT GGC CTT AGT GCA GTG CGG ACC ATG 534 Arg His Leu Pro Glu Lys Gln Asn Gly Leu Ser Ala Val Arg Thr Met 150 155 160 165 GAG GCG TTC CAC TTT GTC AGC TAT GTG CCT ATC ACA GGC CGG CTC TTT 582 Glu Ala Phe His Phe Val Ser Tyr Val Pro Ile Thr Gly Arg Leu Phe 170 175 180 GAG CTG GAT GGG CTG AAG GTC TAC CCC ATT GAC CAT GGG CCC TGG GGG 630 Glu Leu Asp Gly Leu Lys Val Tyr Pro Ile Asp His Gly Pro Trp Gly 185 190 195 GAG GAC GAG GAG TGG ACA GAC AAG GCC CGG CGG GTC ATC ATG GAG CGT 678 Glu Asp Glu Glu Trp Thr Asp Lys Ala Arg Arg Val Ile Met Glu Arg 200 205 210 ATC GGC CTC GCC ACT GCA GGG GAG CCC TAC CAC GAC ATC CGC TTC AAC 726 Ile Gly Leu Ala Thr Ala Gly Glu Pro Tyr His Asp Ile Arg Phe Asn 215 220 225 CTG ATG GCA GTG GTG CCC GAC CGC AGG ATC AAG TAT GAG GCC AGG CTG 774 Leu Met Ala Val Val Pro Asp Arg Arg Ile Lys Tyr Glu Ala Arg Leu 230 235 240 245 CAT GTG CTG AAG GTG AAC CGT CAG ACA GTA CTA GAG GCT CTG CAG CAG 822 His Val Leu Lys Val Asn Arg Gln Thr Val Leu Glu Ala Leu Gln Gln 250 255 260 CTG ATA AGA GTA ACA CAG CCA GAG CTG ATT CAG ACC CAC AAG TCT CAA 870 Leu Ile Arg Val Thr Gln Pro Glu Leu Ile Gln Thr His Lys Ser Gln 265 270 275 GAG TCA CAG CTG CCT GAG GAG TCC AAG TCA GCC AGC AAC AAG TCC CCG 918 Glu Ser Gln Leu Pro Glu Glu Ser Lys Ser Ala Ser Asn Lys Ser Pro 280 285 290 CTG GTG CTG GAA GCA AAC AGG GCC CCT GCA GCC TCT GAG GGC AAC CAC 966 Leu Val Leu Glu Ala Asn Arg Ala Pro Ala Ala Ser Glu Gly Asn His 295 300 305 ACA GAT GGT GCA GAG GAG GCG GCT GGT TCA TGC GCA CAA GCC CCA TCC 1014 Thr Asp Gly Ala Glu Glu Ala Ala Gly Ser Cys Ala Gln Ala Pro Ser 310 315 320 325 CAC AGC CCT CCC AAC AAA CCC AAG CTA GTG GTG AAG CCT CCA GGC AGC 1062 His Ser Pro Pro Asn Lys Pro Lys Leu Val Val Lys Pro Pro Gly Ser 330 335 340 AGC CTC AAT GGG GTT CAC CCC AAC CCC ACT CCC ATT GTC CAG CGG CTG 1110 Ser Leu Asn Gly Val His Pro Asn Pro Thr Pro Ile Val Gln Arg Leu 345 350 355 CCG GCC TTT CTA GAC AAT CAC AAT TAT GCC AAG TCC CCC ATG CAG GAG 1158 Pro Ala Phe Leu Asp Asn His Asn Tyr Ala Lys Ser Pro Met Gln Glu 360 365 370 GAA GAA GAC CTG GCG GCA GGT GTG GGC CGC AGC CGA GTT CCA GTC CGC 1206 Glu Glu Asp Leu Ala Ala Gly Val Gly Arg Ser Arg Val Pro Val Arg 375 380 385 CCA CCC CAG CAG TAC TCA GAT GAT GAG GAT GAC TAT GAG GAT GAC GAG 1254 Pro Pro Gln Gln Tyr Ser Asp Asp Glu Asp Asp Tyr Glu Asp Asp Glu 390 395 400 405 GAG GAT GAC GTG CAG AAC ACC AAC TCT GCC CTT AGG TAT AAG GGG AAG 1302 Glu Asp Asp Val Gln Asn Thr Asn Ser Ala Leu Arg Tyr Lys Gly Lys 410 415 420 GGA ACA GGG AAG CCA GGG GCA TTG AGC GGT TCT GCT GAT GGG CAA CTG 1350 Gly Thr Gly Lys Pro Gly Ala Leu Ser Gly Ser Ala Asp Gly Gln Leu 425 430 435 TCA GTG CTG CAG CCC AAC ACC ATC AAC GTC TTG GCT GAG AAG CTC AAA 1398 Ser Val Leu Gln Pro Asn Thr Ile Asn Val Leu Ala Glu Lys Leu Lys 440 445 450 GAG TCC CAG AAG GAC CTC TCA ATT CCT CTG TCC ATC AAG ACT AGC AGC 1446 Glu Ser Gln Lys Asp Leu Ser Ile Pro Leu Ser Ile Lys Thr Ser Ser 455 460 465 GGG GCT GGG AGT CCG GCT GTG GCA GTG CCC ACA CAC TCG CAG CCC TCA 1494 Gly Ala Gly Ser Pro Ala Val Ala Val Pro Thr His Ser Gln Pro Ser 470 475 480 485 CCC ACC CCC AGC AAT GAG AGT ACA GAC ACG GCC TCT GAG ATC GGC AGT 1542 Pro Thr Pro Ser Asn Glu Ser Thr Asp Thr Ala Ser Glu Ile Gly Ser 490 495 500 GCT TTC AAC TCG CCA CTG CGC TCG CCT ATC CGC TCA GCC AAC CCG ACG 1590 Ala Phe Asn Ser Pro Leu Arg Ser Pro Ile Arg Ser Ala Asn Pro Thr 505 510 515 CGG CCC TCC AGC CCT GTC ACC TCC CAC ATC TCC AAG GTG CTT TTT GGA 1638 Arg Pro Ser Ser Pro Val Thr Ser His Ile Ser Lys Val Leu Phe Gly 520 525 530 GAG GAT GAC AGC CTG CTG CGT GTT GAC TGC ATA CGC TAC AAC CGT GCT 1686 Glu Asp Asp Ser Leu Leu Arg Val Asp Cys Ile Arg Tyr Asn Arg Ala 535 540 545 GTC CGT GAT CTG GGT CCT GTC ATC AGC ACA GGC CTG CTG CAC CTG GCT 1734 Val Arg Asp Leu Gly Pro Val Ile Ser Thr Gly Leu Leu His Leu Ala 550 555 560 565 GAG GAT GGG GTG CTG AGT CCC CTG GCG CTG ACA GAG GGT GGG AAG GGT 1782 Glu Asp Gly Val Leu Ser Pro Leu Ala Leu Thr Glu Gly Gly Lys Gly 570 575 580 TCC TCG CCC TCC ATC AGA CCA ATC CAA GGC AGC CAG GGG TCC AGC AGC 1830 Ser Ser Pro Ser Ile Arg Pro Ile Gln Gly Ser Gln Gly Ser Ser Ser 585 590 595 CCA GTG GAG AAG GAG GTC GTG GAA GCC ACG GAC AGC AGA GAG AAG ACG 1878 Pro Val Glu Lys Glu Val Val Glu Ala Thr Asp Ser Arg Glu Lys Thr 600 605 610 GGG ATG GTG AGG CCT GGC GAG CCC TTG AGT GGG GAG AAA TAC TCA CCC 1926 Gly Met Val Arg Pro Gly Glu Pro Leu Ser Gly Glu Lys Tyr Ser Pro 615 620 625 AAG GAG CTG CTG GCA CTG CTG AAG TGT GTG GAG GCT GAG ATT GCA AAC 1974 Lys Glu Leu Leu Ala Leu Leu Lys Cys Val Glu Ala Glu Ile Ala Asn 630 635 640 645 TAT GAG GCG TGC CTC AAG GAG GAG GTA GAG AAG AGG AAG AAG TTC AAG 2022 Tyr Glu Ala Cys Leu Lys Glu Glu Val Glu Lys Arg Lys Lys Phe Lys 650 655 660 ATT GAT GAC CAG AGA AGG ACC CAC AAC TAC GAT GAG TTC ATC TGC ACC 2070 Ile Asp Asp Gln Arg Arg Thr His Asn Tyr Asp Glu Phe Ile Cys Thr 665 670 675 TTT ATC TCC ATG CTG GCT CAG GAA GGC ATG CTG GCC AAC CTA GTG GAG 2118 Phe Ile Ser Met Leu Ala Gln Glu Gly Met Leu Ala Asn Leu Val Glu 680 685 690 CAG AAC ATC TCC GTG CGG CGG CGC CAA GGG GTC AGC ATC GGC CGG CTC 2166 Gln Asn Ile Ser Val Arg Arg Arg Gln Gly Val Ser Ile Gly Arg Leu 695 700 705 CAC AAG CAG CGG AAG CCT GAC CGG CGG AAA CGC TCT CGC CCC TAC AAG 2214 His Lys Gln Arg Lys Pro Asp Arg Arg Lys Arg Ser Arg Pro Tyr Lys 710 715 720 725 GCC AAG CGC CAG TGAGGACTGC TGGCCCTGAC TCTGCAGCCC ACTCTTGCCG 2266 Ala Lys Arg Gln TGTGGCCCTC ACCAGGGTCC TTCCCTGCCC CACTTCCCCT TTTCCCAGTA TTACTGAATA 2326 GTCCCAGCTG GAGAGTCCAG GCCCTGGGAA TGGGAGGAAC CAGGCCACAT TCCTTCCATC 2386 GTGCCCTGAG GCCTGACACG GCAGATCAGC CCCATAGTGC TCAGGAGGCA GCATCTGGAG 2446 TTGGGGCACA GCGAGGTACT GCAGCTTCCT CCACAGCCGG CTGTGGAGCA GCAGGACCTG 2506 GCCCTTCTGC CTGGGCAGCA GAATATATAT TTTACCTATC AGAGACATCT ATTTTTCTGG 2566 GCTCCAACCC AACATGCCAC CATGTTGACA TAAGTTCCTA CCTGACTATG CTTTCTCTCC 2626 TAGGAGCTGT CCTGGTGGGC CCAGGTCCTT GTATCATCCA CGGTCCCAAC TACAGGGTCC 2686 TAGCTGGGGG CCTGGGTGGG CCCTGGGCTC TGGGCCCTGC TGCTCTAGCC CCAGCCACCA 2746 GCCTGTCCCT GTTGTAAGGA AGCCAGGTCT TCTCTCTTCA TTCCTCTTAG GAGAGTGCCA 2806 AACTCAGGGA CCCAGCACTG GGCTGGGTTG GGAGTAGGGT GTCCCAGTGG GGTTGGGGTG 2866 AGCAGGCTGC TGGGATCCCA TGGCCTGAGC AGAGCATGTG GGAACTGTTC AGTGGCCTGT 2926 GAACTGTCTT CCTTGTTCTA GCCAGGCTGT TCAAGACTGC TCTCCATAGC AAGGTTCTAG 2986 GGCTCTTCGC CTTCAGTGTT GTGGCCCTAG CTATGGGCCT AAATTGGGCT CTAGGTCTCT 3046 GTCCCTGGCG CTTGAGGCTC AGAAGAGCCT CTGTCCAGCC CCTCAGTATT ACCATGTCTC 3106 CCTCTCAGGG GTAGCAGAGA CAGGGTTGCT TATAGGAAGC TGGCACCACT CAGCTCTTCC 3166 TGCTACTCCA GTTTCCTCAG CCTCTGCAAG GCACTCAGGG TGGGGGACAG CAGGATCAAG 3226 ACAACCCGTT GGAGCCCCTG TGTTCCAGAG GACCTGATGC CAAGGGGTAA TGGGCCCAGC 3286 AGTGCCTCTG GAGCCCAGGC CCCAACACAG CCCCATGGCC TCTCCAGATG GCTTTGAAAA 3346 GGTGATCCAA CAGGCCCCTT TATCTGTACA TAGTGACTGA GTGGGGGGTG CTGGCAAGTG 3406 TGGCACTCCT CTGGGCTGAG CACAGCTTGA CCCCTCTAGC CCCTGTAAAA CTGGATCAAT 3466 GAATGAATAA AACTCTCCTA AGATCTCCTG AGAAAAAAAA AAAAAAAAAG G 3517 729 amino acids amino acid linear protein not provided 2 Met Asn Lys Gly Trp Leu Glu Leu Glu Ser Asp Pro Gly Leu Phe Thr 1 5 10 15 Leu Leu Val Glu Asp Phe Gly Val Lys Gly Val Gln Val Glu Glu Ile 20 25 30 Tyr Asp Leu Gln Ser Lys Cys Gln Gly Pro Val Tyr Gly Phe Ile Phe 35 40 45 Leu Phe Lys Trp Ile Glu Glu Arg Arg Ser Arg Arg Lys Val Ser Thr 50 55 60 Leu Val Asp Asp Thr Ser Val Ile Asp Asp Asp Ile Val Asn Asn Met 65 70 75 80 Phe Phe Ala His Gln Leu Ile Pro Asn Ser Cys Ala Thr His Ala Leu 85 90 95 Leu Ser Val Leu Leu Asn Cys Ser Ser Val Asp Leu Gly Pro Thr Leu 100 105 110 Ser Arg Met Lys Asp Phe Thr Lys Gly Phe Ser Pro Glu Ser Lys Gly 115 120 125 Tyr Ala Ile Gly Asn Ala Pro Glu Leu Ala Lys Ala His Asn Ser His 130 135 140 Ala Arg Pro Glu Pro Arg His Leu Pro Glu Lys Gln Asn Gly Leu Ser 145 150 155 160 Ala Val Arg Thr Met Glu Ala Phe His Phe Val Ser Tyr Val Pro Ile 165 170 175 Thr Gly Arg Leu Phe Glu Leu Asp Gly Leu Lys Val Tyr Pro Ile Asp 180 185 190 His Gly Pro Trp Gly Glu Asp Glu Glu Trp Thr Asp Lys Ala Arg Arg 195 200 205 Val Ile Met Glu Arg Ile Gly Leu Ala Thr Ala Gly Glu Pro Tyr His 210 215 220 Asp Ile Arg Phe Asn Leu Met Ala Val Val Pro Asp Arg Arg Ile Lys 225 230 235 240 Tyr Glu Ala Arg Leu His Val Leu Lys Val Asn Arg Gln Thr Val Leu 245 250 255 Glu Ala Leu Gln Gln Leu Ile Arg Val Thr Gln Pro Glu Leu Ile Gln 260 265 270 Thr His Lys Ser Gln Glu Ser Gln Leu Pro Glu Glu Ser Lys Ser Ala 275 280 285 Ser Asn Lys Ser Pro Leu Val Leu Glu Ala Asn Arg Ala Pro Ala Ala 290 295 300 Ser Glu Gly Asn His Thr Asp Gly Ala Glu Glu Ala Ala Gly Ser Cys 305 310 315 320 Ala Gln Ala Pro Ser His Ser Pro Pro Asn Lys Pro Lys Leu Val Val 325 330 335 Lys Pro Pro Gly Ser Ser Leu Asn Gly Val His Pro Asn Pro Thr Pro 340 345 350 Ile Val Gln Arg Leu Pro Ala Phe Leu Asp Asn His Asn Tyr Ala Lys 355 360 365 Ser Pro Met Gln Glu Glu Glu Asp Leu Ala Ala Gly Val Gly Arg Ser 370 375 380 Arg Val Pro Val Arg Pro Pro Gln Gln Tyr Ser Asp Asp Glu Asp Asp 385 390 395 400 Tyr Glu Asp Asp Glu Glu Asp Asp Val Gln Asn Thr Asn Ser Ala Leu 405 410 415 Arg Tyr Lys Gly Lys Gly Thr Gly Lys Pro Gly Ala Leu Ser Gly Ser 420 425 430 Ala Asp Gly Gln Leu Ser Val Leu Gln Pro Asn Thr Ile Asn Val Leu 435 440 445 Ala Glu Lys Leu Lys Glu Ser Gln Lys Asp Leu Ser Ile Pro Leu Ser 450 455 460 Ile Lys Thr Ser Ser Gly Ala Gly Ser Pro Ala Val Ala Val Pro Thr 465 470 475 480 His Ser Gln Pro Ser Pro Thr Pro Ser Asn Glu Ser Thr Asp Thr Ala 485 490 495 Ser Glu Ile Gly Ser Ala Phe Asn Ser Pro Leu Arg Ser Pro Ile Arg 500 505 510 Ser Ala Asn Pro Thr Arg Pro Ser Ser Pro Val Thr Ser His Ile Ser 515 520 525 Lys Val Leu Phe Gly Glu Asp Asp Ser Leu Leu Arg Val Asp Cys Ile 530 535 540 Arg Tyr Asn Arg Ala Val Arg Asp Leu Gly Pro Val Ile Ser Thr Gly 545 550 555 560 Leu Leu His Leu Ala Glu Asp Gly Val Leu Ser Pro Leu Ala Leu Thr 565 570 575 Glu Gly Gly Lys Gly Ser Ser Pro Ser Ile Arg Pro Ile Gln Gly Ser 580 585 590 Gln Gly Ser Ser Ser Pro Val Glu Lys Glu Val Val Glu Ala Thr Asp 595 600 605 Ser Arg Glu Lys Thr Gly Met Val Arg Pro Gly Glu Pro Leu Ser Gly 610 615 620 Glu Lys Tyr Ser Pro Lys Glu Leu Leu Ala Leu Leu Lys Cys Val Glu 625 630 635 640 Ala Glu Ile Ala Asn Tyr Glu Ala Cys Leu Lys Glu Glu Val Glu Lys 645 650 655 Arg Lys Lys Phe Lys Ile Asp Asp Gln Arg Arg Thr His Asn Tyr Asp 660 665 670 Glu Phe Ile Cys Thr Phe Ile Ser Met Leu Ala Gln Glu Gly Met Leu 675 680 685 Ala Asn Leu Val Glu Gln Asn Ile Ser Val Arg Arg Arg Gln Gly Val 690 695 700 Ser Ile Gly Arg Leu His Lys Gln Arg Lys Pro Asp Arg Arg Lys Arg 705 710 715 720 Ser Arg Pro Tyr Lys Ala Lys Arg Gln 725 100 amino acids amino acid unknown protein not provided 3 Met Asp Leu Ser Ala Leu Arg Val Glu Glu Val Gln Asn Val Ile Asn 1 5 10 15 Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cys Leu Glu Leu Ile Lys 20 25 30 Glu Pro Val Ser Thr Lys Cys Asp His Ile Phe Cys Lys Phe Cys Met 35 40 45 Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys 50 55 60 Lys Asn Asp Ile Thr Lys Arg Ser Leu Gln Glu Ser Thr Arg Phe Ser 65 70 75 80 Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cys Ala Phe Gln Leu Asp 85 90 95 Thr Gly Leu Glu 100 100 amino acids amino acid unknown protein not provided 4 Met Asp Leu Ser Ala Val Gln Ile Gln Glu Val Gln Asn Val Leu His 1 5 10 15 Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cys Leu Glu Leu Ile Lys 20 25 30 Glu Pro Val Ser Thr Lys Cys Asp His Ile Phe Cys Lys Phe Cys Met 35 40 45 Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys 50 55 60 Lys Asn Glu Ile Thr Lys Arg Ser Leu Gln Gly Ser Thr Arg Phe Ser 65 70 75 80 Gln Leu Ala Glu Glu Leu Leu Arg Ile Met Ala Ala Phe Glu Leu Asp 85 90 95 Thr Gly Met Gln 100 100 amino acids amino acid unknown protein not provided 5 Met Ala Ser Ser Val Leu Glu Met Ile Lys Glu Glu Val Thr Cys Pro 1 5 10 15 Ile Cys Leu Glu Leu Leu Lys Glu Pro Val Ser Ala Asp Cys Asn His 20 25 30 Ser Phe Cys Arg Ala Cys Ile Thr Leu Asn Tyr Glu Ser Asn Arg Asn 35 40 45 Thr Asp Gly Lys Gly Asn Cys Pro Val Cys Arg Val Pro Tyr Pro Phe 50 55 60 Gly Asn Leu Arg Pro Asn Leu His Val Ala Asn Ile Val Glu Arg Leu 65 70 75 80 Lys Gly Phe Lys Ser Ile Pro Glu Glu Glu Gln Lys Val Asn Ile Cys 85 90 95 Ala Gln His Gly 100 262 amino acids amino acid unknown protein not provided 6 Lys Lys Glu Ile Trp Asn Ser Asp Pro Arg Gly His Glu Gly Pro Gln 1 5 10 15 Pro Ser Pro Thr Pro Ser Asn Glu Ser Thr Asp Thr Ala Ser Glu Ile 20 25 30 Gly Ser Ala Phe Asn Ser Pro Leu Arg Ser Pro Ile Arg Ser Ala Asn 35 40 45 Pro Thr Arg Pro Ser Ser Pro Val Thr Ser His Ile Ser Lys Val Leu 50 55 60 Phe Gly Glu Asp Asp Ser Leu Leu Arg Val Asp Cys Ile Arg Tyr Asn 65 70 75 80 Arg Ala Val Arg Asp Leu Gly Pro Val Ile Ser Thr Gly Leu Leu His 85 90 95 Leu Ala Glu Asp Gly Val Leu Ser Pro Leu Ala Leu Thr Glu Gly Gly 100 105 110 Lys Gly Ser Ser Pro Ser Ile Arg Pro Ile Gln Gly Ser Gln Gly Ser 115 120 125 Ser Ser Pro Val Glu Lys Glu Val Val Glu Ala Thr Asp Ser Arg Glu 130 135 140 Lys Thr Gly Met Val Arg Ser Gly Glu Pro Leu Ser Gly Glu Lys Tyr 145 150 155 160 Ser Pro Lys Glu Leu Leu Ala Leu Leu Lys Cys Val Glu Ala Glu Ile 165 170 175 Ala Asn Tyr Glu Ala Cys Leu Lys Glu Glu Val Glu Lys Arg Lys Lys 180 185 190 Phe Lys Ile Asp Asp Gln Arg Arg Thr His Asn Tyr Asp Glu Phe Ile 195 200 205 Cys Thr Phe Ile Ser Met Leu Ala Gln Glu Gly Met Leu Ala Asn Leu 210 215 220 Val Glu Gln Asn Ile Ser Val Arg Arg Arg Gln Gly Val Ser Ile Gly 225 230 235 240 Arg Leu His Lys Gln Arg Lys Pro Asp Arg Arg Lys Arg Ser Arg Pro 245 250 255 Tyr Lys Ala Lys Arg Gln 260 188 amino acids amino acid unknown protein not provided 7 Gly Gly Ile Asp Trp Ile Pro Gly Tyr Arg Ala Gln Ile Arg Arg Pro 1 5 10 15 Ser Ser Pro Val Thr Ser His Ile Ser Lys Val Leu Phe Gly Glu Asp 20 25 30 Asp Ser Leu Leu Arg Val Asp Cys Ile Arg Tyr Asn Arg Ala Val Arg 35 40 45 Asp Leu Gly Pro Val Ile Ser Thr Gly Leu Leu His Leu Ala Glu Asp 50 55 60 Gly Val Leu Ser Pro Leu Ala Leu Thr Glu Gly Gly Lys Gly Ser Ser 65 70 75 80 Pro Ser Thr Arg Ser Ser Gln Gly Ser Gln Gly Ser Ser Gly Leu Glu 85 90 95 Glu Lys Glu Val Val Glu Val Thr Glu Ser Arg Asp Lys Pro Gly Leu 100 105 110 Asn Arg Ser Ser Glu Pro Leu Ser Gly Glu Lys Tyr Ser Pro Lys Ile 115 120 125 Asp Asp Gln Arg Arg Thr His Asn Tyr Asp Glu Phe Ile Cys Thr Phe 130 135 140 Ile Ser Met Leu Ala Gln Glu Gly Met Leu Ala Asn Leu Val Glu Gln 145 150 155 160 Asn Ile Ser Val Arg Arg Arg Gln Gly Val Ser Ile Gly Arg Leu His 165 170 175 Lys Gln Arg Lys Pro Asp Arg Arg Met Ser Gly Arg 180 185 161 amino acids amino acid unknown protein not provided 8 Gly Gly Ile Asp Trp Ile Pro Gly Val Arg Ala Gln Ile Arg Pro Ile 1 5 10 15 Ser Ser Ser Ser Pro Ser Thr Arg Ser Ser Gln Gly Ser Gln Gly Ser 20 25 30 Ser Gly Leu Glu Glu Lys Glu Val Val Glu Val Thr Glu Ser Arg Asp 35 40 45 Lys Pro Gly Leu Asn Arg Ser Ser Glu Pro Leu Ser Gly Glu Lys Tyr 50 55 60 Ser Pro Lys Glu Leu Leu Ala Leu Leu Lys Cys Ala Glu Ala Glu Ile 65 70 75 80 Ala Asn Tyr Glu Ala Cys Leu Lys Glu Glu Val Glu Lys Arg Lys Lys 85 90 95 Phe Lys Ile Asp Asp Gln Arg Arg Thr His Asn Tyr Asp Glu Phe Ile 100 105 110 Cys Thr Phe Ile Ser Met Leu Ala Gln Glu Gly Met Leu Ala Asn Leu 115 120 125 Val Glu Gln Asn Ile Ser Val Arg Arg Arg Gln Gly Val Ser Ile Gly 130 135 140 Arg Leu His Lys Gln Arg Lys Pro Asp Arg Arg Lys Arg Ile Ser Gly 145 150 155 160 Arg 149 amino acids amino acid unknown protein not provided 9 Gly Gly Ile Asp Trp Ile Pro Gly Tyr Arg Ala Gln Ile Arg Pro Ile 1 5 10 15 Ser Ser Gly Leu Glu Glu Lys Glu Val Val Glu Val Thr Glu Ser Arg 20 25 30 Asp Lys Pro Gly Leu Asn Arg Ser Ser Glu Pro Leu Ser Gly Glu Lys 35 40 45 Tyr Ser Pro Lys Glu Leu Leu Ala Leu Leu Lys Cys Val Glu Ala Glu 50 55 60 Ile Ala Asn Tyr Glu Ala Cys Leu Lys Glu Glu Val Glu Lys Arg Lys 65 70 75 80 Lys Phe Lys Ile Asp Asp Gln Arg Arg Thr His Asn Tyr Asp Glu Phe 85 90 95 Ile Cys Thr Phe Ile Ser Met Leu Ala Gln Glu Gly Met Leu Ala Asn 100 105 110 Leu Val Glu Gln Asn Ile Ser Val Arg Arg Arg Gln Gly Val Ser Ile 115 120 125 Gly Arg Leu His Lys Gln Arg Lys Pro Asp Arg Arg Lys Arg Ser Glu 130 135 140 Arg Pro Ile Asp Arg 145 80 base pairs nucleic acid single unknown other nucleic acid not provided 10 ATGGACCTGT CTGCTCTGCG TGTTGAAGAA GTTCAAAACG TTATCAACGC TATGCAAAAG 60 ATCCTGGAAT GTCCAATCTG 80 101 base pairs nucleic acid single unknown other nucleic acid not provided 11 GGTTCAGCAG CTTCAGCATA CAGAACTTAC AGAAGATGTG GTCACACTTA GTGGAAACTG 60 GTTCCTTGAT CAGTTCCAGA CAGATTGGAC ATTCCAGGAT C 101 102 base pairs nucleic acid single unknown other nucleic acid not provided 12 GTATGCTGAA GCTGCTGAAC CAAAAGAAGG GTCCATCTCA ATGTCCACTG TGTAAGAACG 60 ACATCACTAA GCGTTCTCTG CAAGAATCTA CTCGTTTCTC TC 102 81 base pairs nucleic acid single unknown other nucleic acid not provided 13 TTCCAGACCA GTGTCCAGCT GGAAAGCACA GATGATCTTC AGCAGTTCTT CAACCAGTTG 60 AGAGAAACGA GTAGATTCTT G 81 31 base pairs nucleic acid single unknown other nucleic acid not provided 14 GCTAGAATTC ACCATGGACC TGTCTGCTCT G 31 28 base pairs nucleic acid single unknown other nucleic acid not provided 15 GCTAGTCGAC TTCCAGACCA GTGTCCAG 28 247 amino acids amino acid unknown protein not provided 16 Met Gly Lys Lys Ile Met Thr Asp Ala Gly Ser Trp Cys Leu Ile Glu 1 5 10 15 Ser Asp Pro Gly Val Phe Thr Glu Met Leu Arg Gly Phe Gly Val Asp 20 25 30 Gly Leu Gln Val Glu Glu Leu Tyr Ser Leu Asp Asp Asp Lys Ala Met 35 40 45 Thr Arg Pro Thr Tyr Gly Leu Ile Phe Leu Phe Lys Trp Arg Gln Gly 50 55 60 Asp Glu Thr Thr Gly Ile Pro Ser Asp Lys Gln Asn Ile Phe Phe Ala 65 70 75 80 His Gln Thr Ile Gln Asn Ala Cys Ala Thr Gln Ala Leu Ile Asn Leu 85 90 95 Leu Met Asn Val Glu Asp Thr Asp Val Lys Leu Gly Asn Ile Leu Asn 100 105 110 Gln Tyr Lys Glu Phe Ala Ile Asp Leu Asp Pro Asn Thr Arg Gly His 115 120 125 Cys Leu Ser Asn Ser Glu Glu Ile Arg Thr Val His Asn Ser Phe Ser 130 135 140 Arg Gln Thr Leu Phe Glu Leu Asp Ile Lys Gly Gly Glu Ser Glu Asp 145 150 155 160 Asn Tyr His Phe Val Thr Tyr Val Pro Ile Gly Asn Lys Val Tyr Glu 165 170 175 Leu Asp Gly Leu Arg Glu Leu Pro Leu Glu Val Ala Glu Phe Gln Lys 180 185 190 Glu Gln Asp Trp Ile Glu Ala Ile Lys Pro Val Ile Gln Gln Arg Met 195 200 205 Gln Lys Tyr Ser Glu Gly Glu Ile Thr Phe Asn Leu Met Ala Leu Val 210 215 220 Pro Asn Arg Lys Gln Lys Leu Gln Glu Met Met Glu Asn Leu Ile Gln 225 230 235 240 Ala Asn Glu Asn Asn Glu Leu 245 223 amino acids amino acid unknown protein not provided 17 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val 1 5 10 15 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu 20 25 30 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala 35 40 45 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys 50 55 60 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr 65 70 75 80 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile 85 90 95 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser 100 105 110 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp 115 120 125 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp 130 135 140 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 His Phe Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp 165 170 175 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr 180 185 190 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu 195 200 205 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala 210 215 220 230 amino acids amino acid unknown protein not provided 18 Met Glu Gly Gln Arg Trp Leu Pro Leu Glu Ala Asn Pro Glu Val Thr 1 5 10 15 Asn Gln Phe Leu Lys Gln Leu Gly Leu His Pro Asn Trp Gln Phe Val 20 25 30 Asp Val Tyr Gly Met Asp Pro Glu Leu Leu Ser Met Val Pro Arg Pro 35 40 45 Val Cys Ala Val Leu Leu Leu Phe Pro Ile Thr Glu Lys Tyr Glu Val 50 55 60 Phe Arg Thr Glu Glu Glu Glu Lys Ile Lys Ser Gln Gly Gln Asp Val 65 70 75 80 Thr Ser Ser Val Tyr Phe Met Lys Gln Thr Ile Ser Asn Ala Cys Gly 85 90 95 Thr Ile Gly Leu Ile His Ala Ile Ala Asn Asn Lys Asp Lys Met His 100 105 110 Phe Glu Ser Gly Ser Thr Leu Lys Lys Phe Leu Glu Glu Ser Val Ser 115 120 125 Met Ser Pro Glu Glu Arg Ala Arg Tyr Leu Glu Asn Tyr Asp Ala Ile 130 135 140 Arg Val Thr His Glu Thr Ser Ala His Glu Gly Gln Thr Glu Ala Pro 145 150 155 160 Ser Ile Asp Glu Lys Val Asp Leu His Phe Ile Ala Leu Val His Val 165 170 175 Asp Gly His Leu Tyr Glu Leu Asp Gly Arg Lys Pro Phe Pro Ile Asn 180 185 190 His Gly Glu Thr Ser Asp Glu Thr Leu Leu Glu Asp Ala Ile Glu Val 195 200 205 Cys Lys Lys Phe Met Glu Arg Asp Pro Asp Glu Leu Arg Phe Asn Ala 210 215 220 Ile Ala Leu Ser Ala Ala 225 230 43 amino acids amino acid unknown protein not provided 19 Leu Leu Arg Cys Ser Arg Cys Thr Asn Ile Leu Arg Glu Pro Val Cys 1 5 10 15 Leu Gly Gly Cys Glu His Ile Phe Cys Ser Asn Cys Val Ser Asp Cys 20 25 30 Ile Gly Thr Gly Cys Pro Val Cys Tyr Thr Pro 35 40 326 amino acids amino acid unknown protein not provided 20 Met Gly Lys Lys Ile Met Thr Asp Ala Gly Ser Trp Cys Leu Ile Glu 1 5 10 15 Ser Asp Pro Gly Val Phe Thr Glu Met Leu Arg Gly Phe Gly Val Asp 20 25 30 Gly Leu Gln Val Glu Glu Leu Tyr Ser Leu Asp Asp Asp Lys Ala Met 35 40 45 Thr Arg Pro Thr Tyr Gly Leu Ile Phe Leu Phe Lys Trp Arg Gln Gly 50 55 60 Asp Glu Thr Thr Gly Ile Pro Ser Asp Lys Gln Asn Ile Phe Phe Ala 65 70 75 80 His Gln Thr Ile Gln Asn Ala Cys Ala Thr Gln Ala Leu Ile Asn Leu 85 90 95 Leu Met Asn Val Glu Asp Thr Asp Val Lys Leu Gly Asn Ile Leu Asn 100 105 110 Gln Tyr Lys Glu Phe Ala Ile Asp Leu Asp Pro Asn Thr Arg Gly His 115 120 125 Cys Leu Ser Asn Ser Glu Glu Ile Arg Thr Val His Asn Ser Phe Ser 130 135 140 Arg Gln Thr Leu Phe Glu Leu Asp Ile Lys Gly Gly Glu Ser Glu Asp 145 150 155 160 Asn Tyr His Phe Val Thr Tyr Val Pro Ile Gly Asn Lys Val Tyr Glu 165 170 175 Leu Asp Gly Leu Arg Glu Leu Pro Leu Glu Val Ala Glu Phe Gln Lys 180 185 190 Glu Gln Asp Trp Ile Glu Ala Ile Lys Pro Val Ile Gln Gln Arg Met 195 200 205 Gln Lys Tyr Ser Glu Gly Glu Ile Thr Phe Asn Leu Met Ala Leu Val 210 215 220 Pro Asn Arg Lys Gln Lys Leu Gln Glu Met Met Glu Asn Leu Ile Gln 225 230 235 240 Ala Asn Glu Asn Asn Glu Leu Glu Glu Gln Ile Ala Asp Leu Asn Lys 245 250 255 Ala Ile Ala Asp Glu Asp Tyr Lys Met Glu Met Tyr Arg Lys Glu Asn 260 265 270 Asn Arg Arg Arg His Asn Tyr Thr Pro Phe Val Ile Glu Leu Met Lys 275 280 285 Ile Leu Ala Lys Glu Gly Lys Leu Val Gly Leu Val Asp Asn Ala Tyr 290 295 300 Gln Ala Ala Lys Glu Lys Ser Lys Leu Asn Thr Asp Ile Thr Lys Leu 305 310 315 320 Glu Leu Lys Arg Lys Gln 325 227 amino acids amino acid unknown protein not provided 21 Met Leu Thr Trp Thr Pro Leu Glu Ser Asn Pro Glu Val Leu Thr Lys 1 5 10 15 Tyr Ile His Lys Leu Ala Val Ser Pro Ala Trp Ser Val Thr Asp Val 20 25 30 Ile Gly Leu Glu Asp Asp Thr Leu Glu Trp Ile Pro Arg Pro Val Lys 35 40 45 Ala Phe Ile Leu Leu Phe Pro Cys Ser Glu Thr Tyr Glu Lys His Arg 50 55 60 Thr Glu Glu His Asp Arg Ile Lys Glu Val Glu Glu Gln His Pro Glu 65 70 75 80 Asp Leu Phe Tyr Met Arg Gln Phe Thr His Asn Ala Cys Gly Thr Val 85 90 95 Ala Leu Ile His Ser Val Ala Asn Asn Lys Glu Val Asp Ile Asp Arg 100 105 110 Gly Val Leu Lys Asp Phe Leu Glu Lys Thr Ala Ser Leu Ser Pro Glu 115 120 125 Glu Arg Gly Arg Ala Leu Glu Lys Asp Glu Lys Phe Thr Ala Asp His 130 135 140 Glu Ala Leu Ala Gln Glu Gly Gln Thr Asn Ala Ala Asn His Glu Lys 145 150 155 160 Val Ile His His Phe Ile Ala Leu Val Asn Lys Glu Gly Thr Leu Tyr 165 170 175 Glu Leu Asp Gly Arg Lys Ser Phe Pro Ile Lys His Gly Pro Thr Ser 180 185 190 Glu Glu Thr Phe Val Lys Asp Ala Ala Lys Val Cys Lys Glu Phe Met 195 200 205 Ala Arg Asp Pro Asn Glu Val Arg Phe Thr Val Leu Ala Leu Thr Ala 210 215 220 Ala Gln Gln 225 236 amino acids amino acid unknown protein not provided 22 Met Ser Gly Glu Asn Arg Ala Val Val Pro Ile Glu Ser Asn Pro Glu 1 5 10 15 Val Phe Thr Asn Phe Ala His Lys Leu Gly Leu Lys Asn Glu Trp Ala 20 25 30 Tyr Phe Asp Ile Tyr Ser Leu Thr Glu Pro Glu Leu Leu Ala Phe Leu 35 40 45 Pro Arg Pro Val Lys Ala Ile Val Leu Leu Phe Pro Ile Asn Glu Asp 50 55 60 Arg Lys Ser Ser Thr Ser Gln Gln Ile Thr Ser Ser Tyr Asp Val Ile 65 70 75 80 Trp Phe Lys Gln Ser Val Lys Asn Ala Cys Gly Leu Tyr Ala Ile Leu 85 90 95 His Ser Leu Ser Asn Asn Gln Ser Leu Leu Glu Pro Gly Ser Asp Leu 100 105 110 Asp Asn Phe Leu Lys Ser Gln Ser Asp Thr Ser Ser Ser Lys Asn Arg 115 120 125 Phe Asp Asp Val Thr Thr Asp Gln Phe Val Leu Asn Val Ile Lys Glu 130 135 140 Asn Val Gln Thr Phe Ser Thr Gly Gln Ser Glu Ala Pro Glu Ala Thr 145 150 155 160 Ala Asp Thr Asn Leu His Tyr Ile Thr Tyr Val Glu Glu Asn Gly Gly 165 170 175 Ile Phe Glu Leu Asp Gly Arg Asn Leu Ser Gly Pro Leu Tyr Leu Gly 180 185 190 Lys Ser Asp Pro Thr Ala Thr Asp Leu Ile Glu Gln Glu Leu Val Arg 195 200 205 Val Arg Val Ala Ser Tyr Met Glu Asn Ala Asn Glu Glu Asp Val Leu 210 215 220 Asn Phe Ala Met Leu Gly Leu Gly Pro Asn Trp Glu 225 230 235 230 amino acids amino acid unknown protein not provided 23 Met Glu Gly Gln Arg Trp Leu Pro Leu Glu Ala Asn Pro Glu Val Thr 1 5 10 15 Asn Gln Phe Leu Lys Gln Leu Gly Leu His Pro Asn Trp Gln Phe Val 20 25 30 Asp Val Tyr Gly Met Asp Pro Glu Leu Leu Ser Met Val Pro Arg Pro 35 40 45 Val Cys Ala Val Leu Leu Leu Phe Pro Ile Thr Glu Lys Tyr Glu Val 50 55 60 Phe Arg Thr Glu Glu Glu Glu Lys Ile Lys Ser Gln Gly Gln Asp Val 65 70 75 80 Thr Ser Ser Val Tyr Phe Met Lys Gln Thr Ile Ser Asn Ala Cys Gly 85 90 95 Thr Ile Gly Leu Ile His Ala Ile Ala Asn Asn Lys Asp Lys Met His 100 105 110 Phe Glu Ser Gly Ser Thr Leu Lys Lys Phe Leu Glu Glu Ser Val Ser 115 120 125 Met Ser Pro Glu Glu Arg Ala Arg Tyr Leu Glu Asn Tyr Asp Ala Ile 130 135 140 Arg Val Thr His Glu Thr Ser Ala His Glu Gly Gln Thr Glu Ala Pro 145 150 155 160 Ser Ile Asp Glu Lys Val Asp Leu His Phe Ile Ala Leu Val His Val 165 170 175 Asp Gly His Leu Tyr Glu Leu Asp Gly Arg Lys Pro Phe Pro Ile Asn 180 185 190 His Gly Glu Thr Ser Asp Glu Thr Leu Leu Glu Asp Ala Ile Glu Val 195 200 205 Cys Lys Lys Phe Met Glu Arg Asp Pro Asp Glu Leu Arg Phe Asn Ala 210 215 220 Ile Ala Leu Ser Ala Ala 225 230 223 amino acids amino acid unknown protein not provided 24 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val 1 5 10 15 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu 20 25 30 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala 35 40 45 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys 50 55 60 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr 65 70 75 80 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile 85 90 95 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser 100 105 110 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp 115 120 125 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp 130 135 140 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 His Phe Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp 165 170 175 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr 180 185 190 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu 195 200 205 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala 210 215 220 24 base pairs nucleic acid single unknown other nucleic acid not provided 25 CCATCTCAAG GTCCACTGTG TAAG 24 24 base pairs nucleic acid single unknown other nucleic acid YES not provided 26 CTTACACAGT GGACCTTGAG ATGG 24 27 base pairs nucleic acid single unknown other nucleic acid not provided 27 CAATGTCCAC TGGGTAAGAA CGACATC 27 27 base pairs nucleic acid single unknown other nucleic acid YES not provided 28 GATGTCGTTC TTACCCAGTG GACATTG 27 79 base pairs nucleic acid single unknown other nucleic acid not provided 29 GCATGGATCC TCAAACCTTG TGCAGGCAGG TACCCTGGTC AACAGGAGAC AGGTGGGAAA 60 CCAGGATCTT TTGCATAGC 79 23 base pairs nucleic acid single unknown other nucleic acid not provided 30 CCGATGCCCT TGGAATTGAC GAG 23 25 base pairs nucleic acid single unknown other nucleic acid not provided 31 CGATGAATTC GAGCTAGCTT CTATC 25 32 base pairs nucleic acid single unknown other nucleic acid not provided 32 GCATGAATTC TCAGCTCCGG CGCACTGAGA TG 32 33 base pairs nucleic acid single unknown other nucleic acid not provided 33 GCATGAATTC TCAAGCCAGC ATGGATATGA AGG 33 32 base pairs nucleic acid single unknown other nucleic acid not provided 34 GCATGAATTC TCAGTCATCA ATCTTGAACT TC 32 31 base pairs nucleic acid single unknown other nucleic acid not provided 35 GCATGAATTC TCATGCAATC TCGGCTTCTA C 31 33 base pairs nucleic acid single unknown other nucleic acid not provided 36 GCATGGATCC CCAAGATTGA TGACCAGCGA AGG 33 22 base pairs nucleic acid single unknown other nucleic acid not provided 37 GCTGGCCAAC CCGGTGGAAC AG 22 22 base pairs nucleic acid single unknown other nucleic acid YES not provided 38 CTGTTCCACC GGGTTGGCCA GC 22 56 base pairs nucleic acid single unknown other nucleic acid not provided 39 CCTGTTATTA ACCCTCACTA AAGGGAAGGG TACCATGAAT AAGGGCTGGC TGGAGC 56 20 base pairs nucleic acid single unknown other nucleic acid not provided 40 GAAGCGGATG TCGTGGTAGG 20 22 base pairs nucleic acid single unknown other nucleic acid not provided 41 GATGTATATA ACTATCTATT CG 22 34 base pairs nucleic acid single unknown other nucleic acid not provided 42 GCATAGATCT TCACCCCTGG CTGCCTTGGA TTGG 34 20 base pairs nucleic acid single unknown other nucleic acid not provided 43 GAAGCGGATG TCGTGGTAGG 20 22 base pairs nucleic acid single unknown other nucleic acid not provided 44 GATGTATATA ACTATCTATT CG 22 29 base pairs nucleic acid single unknown other nucleic acid not provided 45 CGTAGTCGAC TGTCAGCGCC AGGGGACTC 29 19 base pairs nucleic acid single unknown other nucleic acid not provided 46 CAACCCCACT CCCATTGTC 19 20 base pairs nucleic acid single unknown other nucleic acid YES not provided 47 GAGTTGGTGT TCTGCACGTC 20 

What is claimed is:
 1. A nucleic acid sequence encoding mammalian BRCA1 Associated Protein (BAP-1) comprising nucleotides 40-2226 of SEQ ID NO: 1, or a sequence completely complementary thereto, isolated from cellular materials with which it is naturally associated.
 2. An isolated fragment of a nucleic acid sequence encoding mammalian BRCA1 Associated Protein (BAP-1), wherein said fragment is selected from the group consisting of: (a) an interactive domain, nucleotides 1831 to 2226 of SEQ ID NO: 1; and (b) a sequence completely complementary to the sequence of (a).
 3. The sequence according to claim 1 which encodes human BAP-1 amino acid numbers 1 to 729 of SEQ ID NO:
 2. 4. A recombinant molecule including a mammalian nucleic acid sequence encoding a BRCA1 associated protein (BAP-1) or peptide, said nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under the control of regulatory sequences, which permit expression of the BAP-1 protein or peptide in a host cell.
 5. A host cell transformed with the recombinant molecule according to claim
 4. 6. A method of recombinantly expressing BRCA1 associated protein (BAP-1) comprising culturing a recombinant host cell transformed with a nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under conditions which permit expression of BAP-1.
 7. A reagent including an isolated or recombinant nucleic acid sequence selected from the group consisting of: (a) SEQ ID NO: 1; (b) a nucleotide sequence containing nucleotides 40 to 2226 of SEQ ID NO: 1; (c) a nucleotide sequence consisting of a nucleotide sequence encoding amino acids 598 to 729 of SEQ ID NO: 2; (d) a sequence completely complementary to a sequence of (a) through (c); and a detectable label which is bound to said sequence.
 8. A composition comprising an isolated or recombinant nucleic acid molecule selected from the group consisting of (a) a plasmid comprising a nucleic acid sequence encoding a BAP-1 protein or peptide, said nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under the control of regulatory sequences directing expression of the encoded protein or peptide in a host cell; (b) a virus vector comprising a nucleic acid sequence encoding a BAP-1 protein or peptide, said nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under the control of regulatory sequence directing expression of the encoded protein or peptide in a host cell; and a pharmaceutically acceptable carrier.
 9. A recombinant molecule including a nucleic acid sequence selected from the group consisting of (a) SEQ ID NO: 1; (b) a nucleotide sequence containing nucleotides 40 to 2226 of SEQ ID NO: 1; (c) a nucleotide sequence consisting of a nucleotide sequence encoding amino acids 598 to 729 of SEQ ID NO: 2; and (d) a sequence completely complementary to a sequence of (a) through (c); and a detectable label.
 10. A plasmid comprising a nucleic acid sequence encoding a BAP-1 protein or peptide, said nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under the control of regulatory sequences directing expression of the encoded protein or peptide in a host cell.
 11. A virus vector comprising a nucleic acid sequence encoding a BAP-1 protein or peptide, said nucleic acid sequence comprising nucleotides 40-2226 of SEQ ID NO: 1 under the control of regulatory sequence directing expression of the encoded protein or peptide in a host cell. 